Ectopic expression of UBX and ABD-B proteins during Drosophila embryogenesis: competition, not a functional hierarchy, explains phenotypic suppression

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 841-854 ◽  
Author(s):  
M.L. Lamka ◽  
A.M. Boulet ◽  
S. Sakonju
Keyword(s):  
Ectopic Expression ◽  
Homeotic Genes ◽  
Wild Type ◽  
Homeodomain Proteins ◽  
Regulatory Interactions ◽  
M Proteins ◽  
Dose Dependent Fashion ◽  
Functional Hierarchy ◽  
Dose Dependent ◽  
Similar Stage

The Abdominal-B (Abd-B) gene, a member of the bithorax complex (BX-C), specifies the identities of parasegments (PS) 10–14 in Drosophila. Abd-B codes for two structurally related homeodomain proteins, ABD-B m and ABD-B r, that are expressed in PS10-13 and PS14-15, respectively. Although ABD-B m and r proteins have distinct developmental functions, ectopic expression of either protein during embryogenesis induces the development of filzkorper and associated spiracular hairs, structures normally located in PS13, at ectopic sites in the larval thorax and abdomen. These results suggest that other parasegmental differences contribute to the phenotype specified by ABD-B r activity in PS14. Both ABD-B m and r repress the expression of other homeotic genes, such as Ubx and abd-A, in PS10-14. However, the importance of these and other cross-regulatory interactions among homeotic genes has been questioned. Since ectopic UBX protein apparently failed to transform abdominal segments, Gonzalez-Reyes et al. (Gonzalez-Reyes, A., Urquia, N., Gehring, W.J., Struhl, G. and Morata, G. (1990). Nature 344, 78–80) proposed a functional hierarchy in which ABD-A and ABD-B activities override UBX activity. We tested this model by expressing UBX and ABD-B m proteins ectopically in wild-type and BX-C-deficient embryos. Ectopic ABD-B m does not prevent transformations induced by ectopic UBX. Instead, ectopic UBX and ABD-B m proteins compete for the specification of segmental identities in a dose-dependent fashion. Our results support a quantitative competition among the homeotic proteins rather than the existence of a strict functional hierarchy. Therefore, we suggest that cross-regulatory interactions are not irrelevant but are important for repressing the expression of competing homeotic proteins. To explain the apparent failure of ectopic UBX to transform the abdominal segments, we expressed UBX at different times during embryonic development. Our results show that ectopic UBX affects abdominal cuticular identities if expressed during early stages of embryogenesis. In later embryonic stages, abdominal segments become resistant to transformation by ectopic UBX while thoracic segments remain susceptible. Head segments also show a similar stage-dependent susceptibility to transformation by ectopic UBX in early embryogenesis but become resistant in later stages. These results suggest that abdominal and head identities are determined earlier than are thoracic identities.

Dorsotonals/homothorax, the Drosophila homologue of meis1, interacts with extradenticle in patterning of the embryonic PNS

Development ◽  
1998 ◽  
Vol 125 (6) ◽  
pp. 1037-1048 ◽  
Author(s):  
E. Kurant ◽  
C.Y. Pai ◽  
R. Sharf ◽  
N. Halachmi ◽  
Y.H. Sun ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Molecular Mechanisms ◽  
Normal Development ◽  
Protein A ◽  
Ectopic Expression ◽  
Homeotic Genes ◽  
Wild Type ◽  
Protein Activity ◽  
Low Levels ◽  
Restricted Pattern

The homeotic genes of the bithorax complex are required, among other things, for establishing the patterns of sensory organs in the embryonic peripheral nervous system (PNS). However, the molecular mechanisms by which these genes affect pattern formation in the PNS are not understood and other genes that function in this pathway are not characterized. Here we report the phenotypic and molecular analysis of one such gene, homothorax (hth; also named dorsotonals). Mutations in the hth gene seem to alter the identity of the abdominal chordotonal neurons, which depend on Abd-A for their normal development. However, these mutations do not alter the expression of the abd-A gene, suggesting that hth may be involved in modulating abd-A activity. We have generated multiple mutations in the hth locus and cloned the hth gene. hth encodes a homeodomain-containing protein that is most similar to the murine proto-oncogene meis1. The hth gene is expressed throughout embryonic development in a spatially restricted pattern, which is modulated in abdominal segments by abd-A and Ubx. The spatial distribution of the HTH protein during embryonic development is very similar to the distribution of the Extradenticle (EXD) protein, a known modulator of homeotic gene activity. Here we show that the PNS phenotype of exd mutant embryos is virtually indistinguishable from that of hth mutant embryos and does not simply follow the homeotic transformations observed in the epidermis. We also show that the HTH protein is present in extremely low levels in embryos lacking exd activity as compared to wild-type embryos. In contrast, the EXD protein is present in fairly normal levels in hth mutant embryos, but fails to accumulate in nuclei and remains cytoplasmic. Ectopic expression of hth can drive ectopic nuclear localization of EXD. Based on our observations we propose that the genetic interactions between hth and exd serve as a novel mechanism for regulating homeotic protein activity in embryonic PNS development.

scholarly journals Effect of abx, bx and pbx mutations on expression of homeotic genes in Drosophila larvae.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 899-908 ◽  
Author(s):  
J W Little ◽  
C A Byrd ◽  
D L Brower
Keyword(s):  
Ectopic Expression ◽  
Wing Disc ◽  
Homeotic Genes ◽  
Wild Type ◽  
Posterior Compartment ◽  
Altered Expression ◽  
Anterior Compartment ◽  
Regulatory Domains ◽  
Wild Type Phenotype ◽  
Drosophila Larvae

Abstract We have examined the patterns of expression of the homeotic gene Ubx in imaginal discs of Drosophila larvae carrying mutations in the abx, bx and pbx regulatory domains. In haltere discs, all five bx insertion mutations examined led to a general reduction in Ubx expression in the anterior compartment; for a given allele, the strength of the adult cuticle phenotype correlated with the degree of Ubx reduction. Deletions mapping near or overlapping the sites of bx insertions, including three abx alleles and the bx34e-prv(bx-prv) allele, showed greatly reduced Ubx expression in parts of the anterior compartment of the haltere disc; however, anterior patches of strong Ubx expression often remained, in highly variable patterns. As expected, the pbx1 mutation led to reduced Ubx expression in the posterior compartment of the haltere disc; surprisingly, pbx1 also led to altered expression of the en protein near the compartment border in the central region of the disc. In the metathoracic leg, all the bx alleles caused extreme reduction in Ubx expression in the anterior regions, with no allele-specific differences. In contrast, abx and bx-prv alleles resulted in patchy anterior reductions in third leg discs. In the larval central nervous system, abx but not bx alleles affected Ubx expression; the bx-prv deletion gave a wild-type phenotype, but it could not fully complement abx mutations. In the posterior wing disc, the bx-prv allele, and to a much lesser extent the bx34e chromosome from which it arose, led to ectopic expression of Ubx. Unlike other grain-of-function mutations in the BX-C, this phenotype appeared to be partially recessive to wild type. Finally, we asked whether the ppx transformation, which results from early lack of Ubx+ function in the mesothorax and is seen in abx animals, is due to ectopic Scr expression. Some mesothoracic leg and wing discs from abx2 larvae displayed ectopic expression of Scr, which was variable in extent but always confined to the posterior compartment.

scholarly journals Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroEL-GroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, inEscherichia coli

1998 ◽  
Vol 64 (5) ◽  
pp. 1694-1699 ◽  
Author(s):  
Kazuyo Nishihara ◽  
Masaaki Kanemori ◽  
Masanari Kitagawa ◽  
Hideki Yanagi ◽  
Takashi Yura
Keyword(s):  
Japanese Cedar ◽  
Wild Type ◽  
E Coli ◽  
Model Protein ◽  
Dose Dependent Fashion ◽  
In The Wild ◽  
Japanese Cedar Pollen ◽  
K 12 ◽  
Cedar Pollen ◽  
Dose Dependent

ABSTRACT Plasmids that can be used for controlled expression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team were constructed in order to facilitate assessment of the effects of these chaperone teams on folding or assembly of recombinant proteins in Escherichia coli. A typical pACYC184-based plasmid which was obtained could express the major DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams from separate promoters when l-arabinose and tetracycline, respectively, were added in a dose-dependent fashion. The model protein used to determine whether this system was useful was an allergen of Japanese cedar pollen, Cryj2, which was unstable when it was produced in E. coli K-12. The effects of chaperone coexpression on the folding, aggregation, and stability of Cryj2 were examined in the wild type and in several mutant bacteria. Coexpression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 without extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroES) or heat shock transcription factor ς32 revealed that both chaperone teams are critically involved in Cryj2 folding but that they are involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of Cryj2 in the absence of ς32 at certain temperatures.

Ectopic expression of wingless in imaginal discs interferes with decapentaplegic expression and alters cell determination

Development ◽  
1996 ◽  
Vol 122 (11) ◽  
pp. 3519-3529 ◽  
Author(s):  
L.A. Johnston ◽  
G. Schubiger
Keyword(s):  
Ectopic Expression ◽  
Imaginal Discs ◽  
Dorsal Side ◽  
Homeotic Genes ◽  
Cell Determination ◽  
Segment Polarity ◽  
High Level ◽  
Dorsal Cells ◽  
Dose Dependent ◽  
Dorsal Pattern

We have expressed the segment polarity gene wingless (wg) ectopically in imaginal discs to examine its regulation of both ventral patterning and transdetermination. By experimentally manipulating the amount of Wg protein, we show that different thresholds of Wg activity elicit different outcomes, which are mediated by regulation of decapentaplegic (dpp) expression and result in alterations in the expression of homeotic genes. A high level of Wg activity leads to loss of all dorsal pattern elements and the formation of a complete complement of ventral pattern elements on the dorsal side of legs, and is correlated with repression of dpp expression. wg expression in dorsal cells of each disc also leads to dose-dependent transdetermination in those cells in homologous discs such as the labial, antennal and leg, but not in cells of dorsally located discs. When dpp expression is repressed by high levels of Wg, transdetermination does not occur, confirming that dpp participates with wg to induce transdetermination. These and other experiments suggest that dorsal expression of wg alters disc patterning and disc cell determination by modulating the expression of dpp. The dose-dependent effects of wg on dpp expression, ventralization of dorsal cells and transdetermination support a model in which wg functions as a morphogen in imaginal discs.

Effects of suboptimal levels of extracellular calcium on the regulation of the cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation in Dictyostelium discoideum

1988 ◽  
Vol 90 (4) ◽  
pp. 691-700 ◽  
Author(s):  
M.B. Coukell ◽  
A.M. Cameron
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Phosphodiesterase Inhibitor ◽  
Wild Type ◽  
Cyclic Amp Phosphodiesterase ◽  
Dose Dependent Fashion ◽  
Inhibitor System ◽  
Membrane Bound ◽  
Dose Dependent

When starved wild-type amoebae of Dictyostelium discoideum were washed and incubated in 1 mM-EGTA, their ability to induce soluble cyclic AMP phosphodiesterase (PD) activity in response to either millimolar cyclic AMP or a series of nanomolar cyclic AMP pulses was reduced by 55–75%. Supplementation of EGTA-treated cells with exogenous Ca2+ stimulated PD induction in a dose-dependent fashion (EC50 = 100–200 nM free extracellular Ca2+), and enzyme production was maximal at about 1 microM free Ca2+. Ca2+ depletion also strongly impaired production of the phosphodiesterase inhibitor (PDI). In contrast, other than delaying their appearance by about 1 h, EGTA had little effect on the induction by cyclic AMP pulses of cell surface markers such as contact sites A and membrane-bound PD activity. Similar changes in both the soluble and membrane activities were observed with strain NP368, a mutant that overproduces cyclic GMP when stimulated by cyclic AMP. Thus, Ca2+ depletion does not appear to inhibit PD and PDI production by reducing intracellular cyclic GMP. To determine whether Ca2+ depletion alters signal transduction, two mutants that produce the soluble PD activities constitutively were examined. Suboptimal concentrations of free extracellular Ca2+ were found to inhibit PD production in these cells to the same degree and with the same concentration dependence as low Ca2+ inhibited PD induction by cyclic AMP in wild-type cells. These results suggest that Ca2+ depletion by EGTA probably inhibits PD and PDI production indirectly by perturbing an intracellular Ca2+ pool(s) rather than by altering a surface cyclic AMP-receptor-mediated process.

scholarly journals Dual orexin receptor antagonists increase sleep and cataplexy in wild type mice

SLEEP ◽  
2019 ◽  
Vol 43 (6) ◽  
Author(s):  
Carrie E Mahoney ◽  
Takatoshi Mochizuki ◽  
Thomas E Scammell
Keyword(s):  
Rem Sleep ◽  
Positive Emotions ◽  
Dark Period ◽  
Nrem Sleep ◽  
Wild Type ◽  
Receptor Antagonists ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Higher Doses ◽  
Desirable Effect

Abstract Orexin receptor antagonists are clinically useful for treating insomnia, but thorough blockade of orexin signaling could cause narcolepsy-like symptoms. Specifically, while sleepiness is a desirable effect, an orexin antagonist could also produce cataplexy, sudden episodes of muscle weakness often triggered by strong, positive emotions. In this study, we examined the effects of dual orexin receptor antagonists (DORAs), lemborexant (E2006) and almorexant, on sleep–wake behavior and cataplexy during the dark period in wild-type (WT) mice and prepro-orexin knockout (OXKO) mice. In WT mice, lemborexant at 10 and 30 mg/kg quickly induced NREM sleep in a dose-dependent fashion. In contrast, lemborexant did not alter sleep–wake behavior in OXKO mice. Under the baseline condition, cataplexy was rare in lemborexant-treated WT mice, but when mice were given chocolate as a rewarding stimulus, lemborexant dose-dependently increased cataplexy. Almorexant produced similar results. Collectively, these results demonstrate that DORAs potently increase NREM and REM sleep in mice via blockade of orexin signaling, and higher doses can cause cataplexy when co-administered with a likely rewarding stimulus.

scholarly journals Bax Loss Impairs Myc-Induced Apoptosis and Circumvents the Selection of p53 Mutations during Myc-Mediated Lymphomagenesis

2001 ◽  
Vol 21 (22) ◽  
pp. 7653-7662 ◽  
Author(s):  
Christine M. Eischen ◽  
Martine F. Roussel ◽  
Stanley J. Korsmeyer ◽  
John L. Cleveland
Keyword(s):  
Transgenic Mice ◽  
Tumor Suppressors ◽  
P53 Mutations ◽  
Wild Type ◽  
Dose Dependent Fashion ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent ◽  
Mdm2 Overexpression ◽  
Selection Of

ABSTRACT The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in Eμ-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in Eμ-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type Eμ-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions inARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in Eμ-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective ofBax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas fromBax-null Eμ-myc transgenics lackedp53 alterations, whereas 27% of the tumors inBax +/− Eμ-myctransgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection ofp53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.

scholarly journals BTN2A, a New Immune-Checkpoint Targeting Vg9Vd2 T Cell Cytotoxicity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1044-1044
Author(s):  
Carla E Cano ◽  
Christine Pasero ◽  
Aude De Gassart ◽  
René Hoet ◽  
Emmanuel Scotet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Cell Cytotoxicity ◽  
Wild Type ◽  
Primary Tumors ◽  
Dose Dependent ◽  
T Cell Cytotoxicity

Background: Anti-tumoral response of Vg9Vd2 T cells requires sensing of phosphoantigens accumulated in malignant cells through binding of butyrophilin 3A(BTN3A). Moreover, an unknown partner located in human Chr6 was shown to be mandatory to BTN3A-mediated Vg9Vd2 T cell activation in murine models. Here, we identified butyrophilin 2A (BTN2A), which is located to Chr6, as a requirement for BTN3A-mediated Vg9Vd2 T cell cytotoxicity against cancer cells. Methods: CRISPR-Cas9-mediated inactivation of BTN2A1/2A2 isoforms was performed in Daudi, K562 and HEK-293T cells. Vg9Vd2 T cells expanded from healthy PBMCs were co-cultured with wild-type or BTN2AKO cells +/- BrHPP (1 µM), HMBPP (0.1 µM) or zoledronate (45 µM), or anti-BTN2A mAb, and Vg9Vd2 T cell degranulation (%CD106ab+ cells), and intracellular TNFa and IFNg assessed after 4h. Mouse T cell hybridoma 53/4 expressing TCRVg9Vd2-MOP were co-cultured overnight with NIH3T3 murine fibroblasts transfected with BTN3A- and/or BTN2A-encoding plasmids +/-HMBPP(10 µM), or increasing doses of HMBPP or anti-BTN3 20.1 mAb. BTN2A transcript expression in normal vs. tumoral tissue was analyzed using GEPIA tool. Anti-BTN2A mAb staining was performed on human samples of primary AML, cervical and pancreatic carcinoma and assessed by flow cytometry. Results: Degranulation and intracellular IFNg/TNFa (n=6) were abolished in Vg9Vd2 T cells co-cultured with BTN2AKO Daudi, K562 and HEK-293T cells compared to wild-type, in all conditions tested including anti-BTN3 20.1. Murine cells do not express no BTN2A1 or BTN3A orthologs and are unable to activate human Vg9Vd2 T cells. Ectopic expression of BTN2A and BTN3A combination but neither BTN2A or BTN3A alone in murine NIH3T3 cells, allows triggering of IL-2 secretion in mouse 53/4-TCRVg9Vd2-MOP reporter cells in presence of HMBPP or 20.1 mAb in dose-dependent manner. Anti-BTN2A mAb was able to suppress Vg9Vd2 T cell degranulation/cytokine secretion against cancer cell lines and activation of mouse 53/4-TCRVg9Vd2-MOP reporter by BTN2A/BTN3A-expressing NIH3T3 in a dose-dependent manner. BTN2A transcript was significantly up-regulated in pancreatic, ovarian and cervical carcinoma vs. normal tissue. Extracellular BTN2A protein was detected in primary hematological and solid tumors. Conclusion: Here, we show that BTN2A is mandatory for BTN3A-mediated function in human Vg9Vd2 T cells. Moreover, concomitant BTN2A and BTN3A expression empowers murine T cells with activation through Vg9Vd2 TCR, opening new roads for mouse models of Vg9Vd2 T cell anti-tumoral responses. We describe an anti-BTN2A able to suppress Vg9Vd2 T cell function, and we show BTN2A expression in primary tumors. These results are relevant for understanding Vg9Vd2 T cell antitumoral immunity triggered by phosphoantigens and amino-bisphosphonates. Disclosures Olive: ImCheck Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; GlaxoSmithKline: Patents & Royalties.

scholarly journals Overexpression of human wild-type FUS causes progressive motor neuron degeneration in an age- and dose-dependent fashion

2012 ◽  
Vol 125 (2) ◽  
pp. 273-288 ◽  
Author(s):  
Jacqueline C. Mitchell ◽  
Philip McGoldrick ◽  
Caroline Vance ◽  
Tibor Hortobagyi ◽  
Jemeen Sreedharan ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Wild Type ◽  
Motor Neuron Degeneration ◽  
Neuron Degeneration ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Human Wild Type

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

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