A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin α-subunit gene

1995 ◽  
Vol 14 (3) ◽  
pp. 313-322 ◽  
Author(s):  
D S Kim ◽  
J H Yoon ◽  
S K Ahn ◽  
K E Kim ◽  
R H Seong ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Nuclear Protein ◽  
Consensus Sequence ◽  
Distal Region ◽  
Proximal Region ◽  
Pituitary Cells ◽  
Maximal Increase ◽  
Α Subunit ◽  
Gel Retardation ◽  
Specific Nuclear Protein

ABSTRACT Our previous studies demonstrated that at least two DNA regions with upstream limits between positions −223 to −190 and positions −151 to −135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (−151 to −135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (−223 to −190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA—protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.

Characterization of Regulatory Elements in the 5′-Upstream Region of the Human Gαq Gene in a Human Megakaryocytic Cell Line.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3534-3534
Author(s):  
Jalagadugula S. Gauthami ◽  
Danny N. Dhanasekharan ◽  
A. Koneti Rao
Keyword(s):  
Protein Binding ◽  
Promoter Activity ◽  
Nuclear Protein ◽  
Consensus Sequence ◽  
Distal Region ◽  
Proximal Region ◽  
Luciferase Reporter ◽  
Positive Region ◽  
Hel Cells ◽  
Megakaryocytic Cell

Abstract GTP-binding protein Gαq plays a major role in platelet signal transduction. We studied the transcriptional regulation of the human Gαq gene in a megakaryocytic cell line, human erythroleukemia (HEL). 10 nM phorbal myristate acetate (PMA) was used to induce megakaryocytic lineage in HEL cells. Western blot analysis on untreated vs PMA-treated HEL lysates showed enhanced Gαq expression with PMA. Firefly luciferase reporter gene constructs carrying various lengths of the Gαq gene upstream promoter region −1/−2727 bp from ATG codon, were transiently transfected into PMA-treated HEL cells. Gαq promoter driven luciferase expression was measured by Dual Luciferase Reporter Assay system (Promega). These studies indicated that the Gαq gene is regulated by positive and negative elements. Two positive regulatory sites were identified in the proximal region (−138/−238 bp) and distal region (−731/−1116 bp); a negative regulatory site was observed further upstream (−1117/−2727 bp). The proximal region −138/−238 was resolved into a repressor (−207/−238) and a positive (−138/−207) site. Consensus sequences for two well recognized megakaryocytic transcription factors (TFs) PU.1 (−225/−230) and GATA-1 (−208/−211) in the repressor site were deleted; this had no effect on promoter activity. The positive region (−138/−207) was further resolved into repressor (−162/−186) and positive (−187/−207) regions by functional studies. In the repressor region a CCACC (−175/−179) consensus motif for Sp/XKLF family of TF was found and deleted; the repressor activity was lost indicating a functional effect of the CCACC-motif. The positive region contained a Sp1/AP2 consensus sites between −152 and −203. Gel shift and super shift experiments on double stranded DNA oligo 1 (−175/−203) and oligo 2 (−152/−174) using HEL extracts demonstrated nuclear protein binding to these regions. However, antibodies against Sp1 and AP2 or the consensus oligos did not alter the binding. Gel shift mutant analysis was performed on both oligos to define the functional sequences mediating the nuclear protein binding. Mutations made at −190/−194, which has a consensus sequence for EGR1/EGR2 and, at −180/−184 and at −156/−160, abolished the protein binding suggesting that they are preferred sequences for DNA-protein interaction. The distal upstream positive region −731/−1116 also revealed positive and negative elements. Mutation in the EGR-2 consensus sequence (−909/−919) decreased the promoter activity to 50% and mutation in the AP2/EGR-1/Sp1 consensus sequence (−885/−893) abolished the promoter activity. Our results suggest that the sequences −156/−160, −180/−184, and −190/−194 in the proximal region, and −885/−893 and −909/−919 in the distal region have a critical role in the transcriptional regulation of Gαq in megakaryocytic cells.

scholarly journals Partial characterization of the human CYP1A1 negatively acting transcription factor and mutational analysis of its cognate DNA recognition sequence.

1995 ◽  
Vol 15 (9) ◽  
pp. 5144-5151 ◽  
Author(s):  
P D Boucher ◽  
M P Piechocki ◽  
R N Hines
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Transcriptional Repression ◽  
Nuclear Protein ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Transient Expression Assay ◽  
Intact Cells ◽  
Specific Nuclear Protein

Previous studies in our laboratory identified a negative regulatory domain in the 5'-flanking region of the human CYP1A1 gene containing two negative regulatory elements (NRE). Characterization of one of these elements revealed three nuclear protein binding regions: a 21-bp palindrome with a point of symmetry at -784 and two guanine- and cytosine-rich elements that flank the palindrome. Functional studies suggested the palindrome is critical for transcriptional repression, whereas the guanine- and cytosine-rich sequences play a secondary role. In this study, the interaction between nuclear proteins and the CYP1A1 NRE was further defined. Electrophoretic mobility shift assays (EMSA) indicated that the NRE -784 palindrome alone, but not the guanine- and cytosine-rich sequences minus the palindrome, was capable of specific nuclear protein binding. Competitive cotransfection experiments confirmed this observation in intact cells. Specific residues important for DNA-protein interactions were identified by site-directed mutagenesis and competitive EMSA. The loss of specific protein binding was also correlated with the loss of negative regulatory activity in a transient-expression assay. Finally, competitive EMSA was performed with consensus oligonucleotides for known transcription factors. An NF-Y consensus sequence efficiently competed with the NRE probe for specific nuclear protein binding. EMSA supershift analyses indicate that a protein immunologically related to NF-YB is part of the specific nuclear protein complex binding the human CYP1A1 NRE. These studies have refined our understanding of the sequences critical for the transcriptional repression of human CYP1A1. To our knowledge, this is also the first report implicating a member of the NF-Y transcription factor family in negative gene regulation.

scholarly journals Intestinal Apolipoprotein A-IV Gene Transcription Is Controlled by Two Hormone-Responsive Elements: A Role for Hepatic Nuclear Factor-4 Isoforms

2005 ◽  
Vol 19 (9) ◽  
pp. 2320-2334 ◽  
Author(s):  
Amena Archer ◽  
Dominique Sauvaget ◽  
Valérie Chauffeton ◽  
Pierre-Etienne Bouchet ◽  
Jean Chambaz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Distal Region ◽  
Proximal Region ◽  
Dominant Negative ◽  
Specific Expression ◽  
Nuclear Factors ◽  
Dominant Negative Form ◽  
Responsive Element ◽  
Hormone Responsive Element

Abstract In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4α and γ. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4α repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4α and γ functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

scholarly journals A Distal Region Involving Hepatocyte Nuclear Factor 4α and CAAT/Enhancer Binding Protein Markedly Potentiates the Protein Kinase A Stimulation of the Glucose-6-Phosphatase Promoter

2005 ◽  
Vol 19 (1) ◽  
pp. 163-174 ◽  
Author(s):  
Amandine Gautier-Stein ◽  
Gilles Mithieux ◽  
Fabienne Rajas
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Binding Sites ◽  
Binding Protein ◽  
Distal Region ◽  
Proximal Region ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Enhancer Binding Protein ◽  
Kinase A

Abstract Glucose-6-phosphatase (Glc6Pase) is the last enzyme of gluconeogenesis and is only expressed in the liver, kidney, and small intestine. In these tissues, the mRNA and its activity are increased when cAMP levels increased (e.g. in fasting or diabetes). We first report that a proximal region (within −200 bp relative to the transcription start site) and a distal region (−694/−500 bp) are both required for a potent cAMP and a protein kinase A (PKA) responsiveness of the Glc6Pase promoter. Using different molecular approaches, we demonstrate that hepatocyte nuclear factor (HNF4α), CAAT/ enhancer-binding protein-α (C/EBPα), C/EBPβ, and cAMP response element-binding protein (CREB) are involved in the potentiated PKA responsiveness: in the distal region, via one HNF4α- and one C/EBP-binding sites, and in the proximal region, via two HNF4α and two CREB-binding sites. We also show that HNF4α, C/EBPα, and C/EBPβ are constitutively bound to the endogenous Glc6Pase gene, whereas CREB and CREB-binding protein (CBP) will be bound to the gene upon stimulation by cAMP. These data strongly suggest that the cAMP responsiveness of the Glc6Pase promoter requires a tight cooperation between a proximal and a distal region, which depends on the presence of several HNF4α-, C/EBP-, and CREB-binding sites, therefore involving an intricate association of hepatic and ubiquitous transcription factors.

Regulatory elements controlling pituitary-specific expression of the human prolactin gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4690-4700
Author(s):  
B Peers ◽  
M L Voz ◽  
P Monget ◽  
M Mathy-Hartert ◽  
M Berwaer ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Distal Region ◽  
Dnase I ◽  
Proximal Region ◽  
Base Pairs ◽  
Specific Expression ◽  
Positive Region ◽  
Dnase I Footprinting ◽  
Human Prolactin ◽  
Flanking Sequences

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

Effect of imposed head vibration on the stability and waveform of flagellar beating in sea urchin spermatozoa

1991 ◽  
Vol 156 (1) ◽  
pp. 63-80 ◽  
Author(s):  
C. Shingyoji ◽  
I. R. Gibbons ◽  
A. Murakami ◽  
K. Takahashi
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Region ◽  
Proximal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Additional Energy ◽  
The Stability ◽  
Cyclic Changes

The heads of live spermatozoa of the sea urchin Hemicentrotus pulcherrimus were held by suction in the tip of a micropipette mounted on a piezoelectric device and vibrated either laterally or axially with respect to the head axis. Within certain ranges of frequency and amplitude, lateral vibration of the pipette brought about a stable rhythmic beating of the flagella in the plane of vibration, with the beat frequency synchronized to the frequency of vibration [Gibbons et al. (1987), Nature 325, 351–352]. The sperm flagella, with an average natural beat frequency of 48 Hz, showed stable beating synchronized to the pipette vibration over a range of 35–90 Hz when the amplitude of vibration was about 20 microns or greater. Vibration frequencies below this range caused instability of the beat plane, often associated with irregularities in beat frequency. Frequencies above about 90 Hz caused irregular asymmetrical flagellar beating with a marked decrease in amplitude of the propagated bends and a skewing of the flagellar axis towards one side; the flagella often stopped in a cane shape. In flagella that were beating stably under imposed vibration, the wavelength was reduced at higher frequencies and increased at lower frequencies. When the beat frequency was equal to or lower than the natural beat frequency, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with beat frequency in both the proximal and distal regions of the flagella. However, at vibration frequencies above the natural beat frequency, the sliding velocity increased with frequency only in the proximal region of the flagellum and remained essentially unchanged in more distal regions. This apparent limit to the velocity of sliding in the distal region may represent an inherent limit in the intrinsic velocity of active sliding, while the faster sliding observed in the proximal region may be a result of passive sliding or elastic distortion of the microtubules induced by the additional energy supplied by the vibrating pipette. Axial vibration with frequencies either close to or twice the natural beat frequency induced cyclic changes in the waveform, compressing and expanding the bends in the proximal region, but did not affect bends in the distal region or alter the beat frequency.

Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals The Distribution and Severity of Corrosion Damage at Eight Distinct Zones of Metallic Femoral Stem Implants

Metals ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 840 ◽  
Author(s):  
Roohollah Milimonfared ◽  
Reza Oskouei ◽  
Mark Taylor ◽  
Lucian Solomon
Keyword(s):  
Spatial Distribution ◽  
Hip Replacement ◽  
Logistic Regression Model ◽  
Statistical Significance ◽  
Femoral Stem ◽  
Corrosion Damage ◽  
Distal Region ◽  
Proximal Region ◽  
Visual Scoring ◽  
Ordinal Logistic Regression Model

Metallic taper junctions of modular total hip replacement implants are analysed for corrosion damage using visual scoring based on different granularity levels that span from analysing the taper holistically to dividing the taper into several distinct zones. This study aims to objectively explore the spatial distribution and the severity of corrosion damage onto the surface of metallic stem tapers. An ordinal logistic regression model was developed to find the odds of receiving a higher score at eight distinct zones of 137 retrieved stem tapers. A method to find the order of damage severity across the eight zones is introduced based on an overall test of statistical significance. The findings show that corrosion at the stem tapers occurred more commonly in the distal region in comparison with the proximal region. Also, the medial distal zone was found to possess the most severe corrosion damage among all the studied eight zones.

Functional morphology of the mouthparts and proventriculus of the rock crab Nectocarcinus tuberculosus (Decapoda: Portunidae)

2003 ◽  
Vol 83 (4) ◽  
pp. 821-834 ◽  
Author(s):  
Indra Raymond Salindeho ◽  
Danielle Jane Johnston
Keyword(s):  
Content Analysis ◽  
Stomach Content ◽  
Stomach Content Analysis ◽  
Distal Region ◽  
Proximal Region ◽  
Rocky Shores ◽  
Brachyuran Crab ◽  
Cardiac Stomach ◽  
Dietary Preferences ◽  
Sea Grass

Mouthpart and proventriculus structure of the brachyuran crab Nectocarcinus tuberculosus was described by scanning electron microscopy and histology and characteristics indicative of dietary preferences were identified. A qualitative stomach content analysis was conducted to verify structural interpretations with respect to diet. The mouthparts and proventriculus of N. tuberculosus have features that are typical of macrophagous crabs and are consistent with a carnivorous diet of hard items. However, the mandibular arrangement indicates that N. tuberculosus is also adapted to ingest soft plant material and fleshy items, revealing that this crab is omnivorous which is consistent with its habitat of rocky shores and sea grass beds. Carnivorous features of the feeding apparatus include large crista dentata on the third maxillipeds that grip food items during ingestion and cuspidate setae on the second maxillipeds and first maxillae. Nectocarcinus tuberculosus has a complex arrangement of proventricular ossicles, 5-denticulated accessory teeth, and a spiny cardio-pyloric valve with rough, calcified protuberances. It has a robust, strongly calcified gastric mill with a prominent anterior cusp and 11 roughly surfaced vertical ridges on each lateral tooth specialized for crushing and grinding. In contrast to true carnivorous crabs, the mandibles in this species are symmetrically arranged, with two sharp cutting edges and no teeth, an arrangement adapted to cut softer plant-like materials and flesh. Structural interpretations with respect to diet were confirmed by stomach content analysis where items identified included gastropods, bivalves and the sea grass Posidonia australis. Functionally, the filtering system in the proventriculus of N. tuberculosus is complex. The cardiac stomach has a well developed ventral filtration system or ‘cardiac filter’ which comprises coarse and fine filters. Different modifications of setation between the distal and proximal regions of the inner valve of the pyloric filter are unique to this crab species, with an intersetule width 600 nm in the distal region and 80–100 nm in the proximal region. This suggests that some differentiation in filtration occurs between regions within the pyloric filter.

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