scholarly journals Synthetic deconstruction of hunchback regulation by Bicoid

2021 ◽  
Author(s):  
Gonçalo Fernandes ◽  
Huy Tran ◽  
Maxime Andrieu ◽  
Youssoupha Diaw ◽  
Carmina Perez Romero ◽  
...  
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Positional Information ◽  
Fruit Fly ◽  
Decay Length ◽  
Activation Process ◽  
Modeling Tools ◽  
Spatial Features ◽  
Correct Location ◽  
Almost All

During development, cell identity is established reproducibly among individuals through the expression of specific genes at the correct time and correct location in space. How genes extract and combine both positional and temporal information from different transcription factor (TF) profiles along polarity axes remain largely unexplored. Here, we showcase the classic hunchback gene in fruit fly embryos, with focus on 3 of its main TFs: Bicoid, Zelda and Hunchback proteins. We constructed a series of synthetic MS2 reporters, where the numbers and combination of binding sites for each TF are varied. Using live imaging of transcription dynamics by these synthetic reporters and modeling tools, we show that i) a Bicoid-only synthetic reporter needs 3 more Bicoid binding sites than found in the hunchback promoter to recapitulates almost all spatial features of early hb expression but takes more time to reach steady state; ii) Hunchback and Zelda binding sites combined with Bicoid sites both reduce the time to reach steady state and increase expression at a different step in the activation process: Zld sites lower the Bicoid threshold required for activation while Hb sites increase the polymerase firing rate and reduce bursting; iii) the shift of the Bicoid-only reporter induced by a reduction by half of Bicoid concentrations indicates that the decay length of the Bicoid activity gradient is lower than the decay length of the Bicoid protein gradient. Altogether, this work indicates that Bicoid is the main source of positional information for hunchback expression and places back the Bicoid system within the physical limits of an equilibrium model.

scholarly journals 3 minutes to precisely measure morphogen concentration

2018 ◽  
Author(s):  
Tanguy Lucas ◽  
Huy Tran ◽  
Carmina Angelica Perez Romero ◽  
Aurélien Guillou ◽  
Cécile Fradin ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Response ◽  
Positional Information ◽  
Fruit Fly ◽  
Fluorescent Labeling ◽  
Hill Coefficient ◽  
Activation Process ◽  
Endogenous Expression ◽  
Time Period ◽  
Zygotic Transcription

AbstractMorphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the main Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Remarkably, despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to measure subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on cooperativity between the 6 known Bicoid binding sites in the hb promoter region and assuming rate limiting concentrations of the Bicoid transcription factor at the boundary is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that additional mechanisms are involved in the steepness of the response.

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

1986 ◽  
Vol 108 (2) ◽  
pp. 267-273 ◽  
Author(s):  
S. Kyakumoto ◽  
R. Kurokawa ◽  
Y. Ohara-Nemoto ◽  
M. Ota
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Binding Sites ◽  
Androgen Receptors ◽  
Dissociation Constants ◽  
Scatchard Analysis ◽  
Male And Female ◽  
Short Period ◽  
Almost All ◽  
Androgen Binding

ABSTRACT Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 °C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5′-phosphate (5 mmol/l) from nuclear fractions with 93–95% efficiency. The exchange of the bound steroids occurred by 24–48 h at 0 °C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0·8 and 0·9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females. J. Endocr. (1986) 108, 267–273

Drug Interactions with Warfarin: Studies with Dichloralphenazone, Chloral Hydrate and Phenazone (Antipyrine)

1971 ◽  
Vol 40 (4) ◽  
pp. 351-364 ◽  
Author(s):  
A. Breckenridge ◽  
M. L'E. Orme ◽  
S. Thorgeirsson ◽  
D. S. Davies ◽  
R. V. Brooks
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Urinary Excretion ◽  
Binding Sites ◽  
Half Life ◽  
Chloral Hydrate ◽  
Maximal Velocity ◽  
Protein Binding Sites ◽  
Drug Oxidation ◽  
State Plasma

1. Administration of dichloralphenazone, a complex of chloral hydrate and phenazone (antipyrine) caused a fall in steady-state plasma warfarin concentration and loss of anticoagulant control in five subjects. 2. This effect of dichloralphenazone is due to stimulation of the drug-oxidizing enzymes of the liver endoplasmic reticulum by antipyrine, the non-hypnotic part of the complex. Administration of antipyrine caused a fall in steady-state plasma warfarin concentration in five subjects, a shortening of the plasma warfarin half-life, with increased urinary excretion of the metabolites of 14C-labelled warfarin in two subjects and increased urinary excretion of 6β-hydroxycortisol which is formed in the liver endoplasmic reticulum. 3. Administration of chloral hydrate, the hypnotic part of dichloralphenazone, caused no change in anticoagulant control but a fall in steady-state plasma warfarin concentration in five subjects. This is due to the accumulation of trichloroacetic acid which displaces warfarin from plasma protein binding sites. 4. Individual differences in the extent of enzyme induction have been shown to be related to the subjects' rates of drug oxidation. 5. In the rat administration of dichloralphenazone and antipyrine, but not chloral hydrate, caused shortening of pentobarbitone sleeping time and of the plasma [14C]pentobarbitone half-life, shortening of the zoxazolamine paralysis time and increase in the maximal velocity of N-demethylation of ethylmorphine.

scholarly journals Fibrinolysis: A Primordial System Linked to the Immune Response

2021 ◽  
Vol 22 (7) ◽  
pp. 3406
Author(s):  
Robert L. Medcalf ◽  
Charithani B. Keragala
Keyword(s):  
Binding Sites ◽  
Defense Mechanisms ◽  
Immune Cell ◽  
Immune Defense ◽  
Surface Proteins ◽  
Cell Behavior ◽  
Phylogenetic Studies ◽  
Blood Clots ◽  
Lower Vertebrates ◽  
Almost All

The fibrinolytic system provides an essential means to remove fibrin deposits and blood clots. The actual protease responsible for this is plasmin, formed from its precursor, plasminogen. Fibrin is heralded as it most renowned substrate but for many years plasmin has been known to cleave many other substrates, and to also activate other proteolytic systems. Recent clinical studies have shown that the promotion of plasmin can lead to an immunosuppressed phenotype, in part via its ability to modulate cytokine expression. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allows plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, a multitude of pathogens can also express their own plasminogen activators, or contain surface proteins that provide binding sites host plasminogen. Plasmin formed under these circumstances also empowers these pathogens to modulate host immune defense mechanisms. Phylogenetic studies have revealed that the plasminogen activating system predates the appearance of fibrin, indicating that plasmin did not evolve as a fibrinolytic protease but perhaps has its roots as an immune modifying protease. While its fibrin removing capacity became apparent in lower vertebrates these primitive under-appreciated immune modifying functions still remain and are now becoming more recognised.

scholarly journals Two Populations of Glucocorticoid Receptor-Binding Sites in the Male Rat Hippocampal Genome

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1832-1844 ◽  
Author(s):  
J. Annelies E. Polman ◽  
E. Ronald de Kloet ◽  
Nicole A. Datson
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Glucocorticoid Receptors ◽  
Full Range ◽  
Male Rat ◽  
Motif Analysis ◽  
Wide Range ◽  
Neuronal Processes ◽  
Almost All

Abstract In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS. Gene ontology clusters involved in neuronal plasticity and other essential neuronal processes were overrepresented among the genes harboring a GBS or located in the vicinity of a GBS. Male adrenalectomized rats were challenged with increasing doses of the GR agonist corticosterone (CORT) ranging from 3 to 3000 μg/kg, resulting in clear differences in the GR-binding profile to individual GBS. Two groups of GBS could be distinguished: a low-CORT group that displayed GR binding across the full range of CORT concentrations, and a second high-CORT group that displayed significant GR binding only after administering the highest concentration of CORT. All validated GBS, in both the low-CORT and high-CORT groups, displayed mineralocorticoid receptor binding, which remained relatively constant from 30 μg/kg CORT upward. Motif analysis revealed that almost all GBS contained a glucocorticoid response element resembling the consensus motif in literature. In addition, motifs corresponding with new potential GR-interacting proteins were identified, such as zinc finger and BTB domain containing 3 (Zbtb3) and CUP (CG11181 gene product from transcript CG11181-RB), which may be involved in GR-dependent transactivation and transrepression, respectively. In conclusion, our results highlight the existence of 2 populations of GBS in the rat hippocampal genome.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4349-4358 ◽  
Author(s):  
J. Charite ◽  
W. de Graaff ◽  
D. Consten ◽  
M.J. Reijnen ◽  
J. Korving ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Positional Information ◽  
Regulatory Elements ◽  
Gene Products ◽  
Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Analytical Study of the Existence of a Hopf Bifurcation in the Tumor Cell Growth Model with Time Delay

2021 ◽  
Vol 3 (1) ◽  
pp. 20-28
Author(s):  
A. Yusnaeni ◽  
Kasbawati Kasbawati ◽  
Toaha Syamsuddin
Keyword(s):  
Differential Equation ◽  
Steady State ◽  
Immune System ◽  
Hopf Bifurcation ◽  
Delay Differential Equation ◽  
Immune Cells ◽  
Steady State Solution ◽  
State Solution ◽  
Activation Process ◽  
Delay Differential

AbstractIn this paper, we study a mathematical model of an immune response system consisting of a number of immune cells that work together to protect the human body from invading tumor cells. The delay differential equation is used to model the immune system caused by a natural delay in the activation process of immune cells. Analytical studies are focused on finding conditions in which the system undergoes changes in stability near a tumor-free steady-state solution. We found that the existence of a tumor-free steady-state solution was warranted when the number of activated effector cells was sufficiently high. By considering the lag of stimulation of helper cell production as the bifurcation parameter, a critical lag is obtained that determines the threshold of the stability change of the tumor-free steady state. It is also leading the system undergoes a Hopf bifurcation to periodic solutions at the tumor-free steady-state solution.Keywords: tumor–immune system; delay differential equation; transcendental function; Hopf bifurcation. AbstrakDalam makalah ini, dikaji model matematika dari sistem respon imun yang terdiri dari sejumlah sel imun yang bekerja sama untuk melindungi tubuh manusia dari invasi sel tumor. Persamaan diferensial tunda digunakan untuk memodelkan sistem kekebalan yang disebabkan oleh keterlambatan alami dalam proses aktivasi sel-sel imun. Studi analitik difokuskan untuk menemukan kondisi di mana sistem mengalami perubahan stabilitas di sekitar solusi kesetimbangan bebas tumor. Diperoleh bahwa solusi kesetimbangan bebas tumor dijamin ada ketika jumlah sel efektor yang diaktifkan cukup tinggi. Dengan mempertimbangkan tundaan stimulasi produksi sel helper sebagai parameter bifurkasi, didapatkan lag kritis yang menentukan ambang batas perubahan stabilitas dari solusi kesetimbangan bebas tumor. Parameter tersebut juga mengakibatkan sistem mengalami percabangan Hopf untuk solusi periodik pada solusi kesetimbangan bebas tumor.Kata kunci: sistem tumor–imun; persamaan differensial tundaan; fungsi transedental; bifurkasi Hopf.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals RETRACTED ARTICLE: The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lin Zhang ◽  
Zhiqiang Song ◽  
Fangfang Li ◽  
Xixi Li ◽  
Haikun Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Drought Stress ◽  
Reporter Gene ◽  
Binding Sites ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Cis Elements ◽  
Gus Reporter ◽  
Almost All

Abstract Background Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences. Results In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5′ deletion mutants of these promoters, it was determined that TaGAPCp2P (− 1312~ − 528) and TaGAPCp3P (− 2049~ − 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (− 1197~ − 635) and TaGAPCp3P (− 1456~ − 1144 and − 718~ − 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (− 1135 and − 985) and TaGAPCp3P (− 1414 and − 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB. Conclusion The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.

scholarly journals A study of the interaction in vitro between type I collagen and a small dermatan sulphate proteoglycan

1988 ◽  
Vol 251 (3) ◽  
pp. 643-648 ◽  
Author(s):  
N Uldbjerg ◽  
C C Danielsen
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Type I Collagen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Side Chains ◽  
Type I ◽  
Dermatan Sulphate ◽  
Collagen Fibrillogenesis ◽  
High Affinity ◽  
Sulphate Proteoglycan

The interaction between a small dermatan sulphate proteoglycan isolated from human uterine cervix and collagen type I from human and rat skin was investigated by collagen-fibrillogenesis experiments. Collagen fibrillogenesis was initiated by elevation of temperature and pH after addition of proteoglycan, chondroitinase-digested proteoglycan or isolated side chains, and monitored by turbidimetry. Collagen-associated and unbound proteoglycan was determined by enzyme-linked immunosorbent assay after aggregation was complete. (1) The binding of proteoglycan to collagen could be explained by the presence of two mutually non-interacting binding sites, with Ka1 = 1.3 x 10(8) M-1 and Ka2 = 1.3 x 10(6) M-1. The number of binding sites per tropocollagen molecule was n1 = 0.11 and n2 = 1.1. The 0.1 high-affinity binding site per tropocollagen molecule indicates that the strong interaction between proteoglycan and collagen results from a concerted action of tropocollagen molecules in fibrils. Digestion of the proteoglycan with chondroitinase ABC did not affect these binding characteristics. (2) Proteoglycan did not affect the rate of fibrillogenesis, but increased the steady-state A400 by up to 90%. This increase was directly proportional to the saturation of the high-affinity type of binding sites. Neither isolated core protein nor isolated side chains induced a similar high increase in steady-state A400. (3) Electron micrographs showed that the fibril diameter was affected only to a minor extent, if at all, by the proteoglycan, whereas bundles of laterally aligned fibrils were common in the presence of proteoglycan. (4) Results obtained with human and rat collagen were similar.

Sign in / Sign up

Export Citation Format

Share Document