scholarly journals Transcription factor p53 exhibits increased binding to the α2-macroglobulin gene promoter and decreased glycosylation in fetal and adult rat liver during the acute-phase response

2008 ◽  
Vol 60 (3) ◽  
pp. 347-353
Author(s):  
Mirjana Mihailovic ◽  
G. Poznanovic ◽  
Svetlana Dinic ◽  
Nevena Grdovic ◽  
Melita Vidakovic ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Acute Phase ◽  
Gene Promoter ◽  
Immunoblot Analysis ◽  
Binding Potential ◽  
Adult Rat ◽  
Α2 Macroglobulin ◽  
Transcription Factor P53 ◽  
Dna Affinity Chromatography

The binding affinity of p53 for the MG promoter was assessed by DNA-affinity chromatography with the extended ?2-macroglobulin (MG) gene promoter (-852/+12) and immunoblot analysis. During the increased MG gene transcription observed in the fetus and the acute-phase (AP) response in both the fetus and the adult, p53 exhibited increased binding to the MG promoter. This increase was accompanied by decreased O-linked N-acetyl glucosamine glycosylation of p53. We suggest that the enzymatic removal of sugar moieties in vivo serves to activate the MG gene promoter binding potential of p53 and its participation in upregulated MG gene transcription.

scholarly journals In vitro transcription from the human A gamma-globin gene promoter

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1344-1350 ◽  
Author(s):  
M Castle ◽  
D O'Neill ◽  
A Bank
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Systematic Analysis ◽  
Gamma Globin ◽  
Octamer Motif

Abstract We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

scholarly journals Postgenomic Analysis of Four Novel Antigens of Group A Streptococcus: Growth Phase-Dependent Gene Transcription and Human Serologic Response

2002 ◽  
Vol 184 (22) ◽  
pp. 6316-6324 ◽  
Author(s):  
Sean D. Reid ◽  
Nicole M. Green ◽  
Gail L. Sylva ◽  
Jovanka M. Voyich ◽  
Elisha T. Stenseth ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Group A Streptococcus ◽  
Immunoblot Analysis ◽  
Surface Expression ◽  
Extracellular Proteins ◽  
Infection Model ◽  
Gene Transcript ◽  
Serum Samples ◽  
Group A

ABSTRACT Analysis of three group A Streptococcus genomes (serotypes M1, M3, and M18) recently identified four previously undescribed genes that encode extracellular proteins. Each of these genes encode proteins with an LPXTG amino acid motif that covalently links many virulence factors produced by gram-positive bacteria to the cell surface. Western immunoblot analysis of serum samples obtained from 80 patients with invasive infections, noninvasive soft tissue infections, pharyngitis, and rheumatic fever indicated that these four proteins are expressed in vivo. However, the level of gene transcript and the time of maximal gene transcription varied in representative serotype M1, M3, and M18 strains. Surface expression of two proteins was confirmed by flow cytometry. Studies using a mouse infection model suggest that antibodies specific for one of the proteins (Spy0843) may contribute to a protective host immune response against a serotype M1 infection. These results are additional evidence that postgenomic strategies provide new ways to identify and investigate novel bacterial proteins that may participate in host-pathogen interactions or serve as targets for therapeutics research.

Regulation of the rat NHE3 gene promoter by sodium butyrate

2001 ◽  
Vol 281 (4) ◽  
pp. G947-G956 ◽  
Author(s):  
Pawel R. Kiela ◽  
Eric R. Hines ◽  
James F. Collins ◽  
Fayez K. Ghishan
Keyword(s):  
Gene Transcription ◽  
Transcriptional Activation ◽  
Hdac Inhibitor ◽  
Promoter Activity ◽  
Actinomycin D ◽  
Trichostatin A ◽  
Gene Promoter ◽  
Short Chain Fatty Acids

Short-chain fatty acids, and especially butyrate (NaB), stimulate sodium and water absorption by inducing colonic Na+/H+ exchange (NHE). NaB induces NHE3 activity and protein and mRNA expression both in vivo and in vitro. NaB, as a histone deacetylase (HDAC) inhibitor, regulates gene transcription. We therefore studied whether NaB regulates transcription of the rat NHE3 promoter in transiently transfected Caco-2 cells. NaB (5 mM) strongly stimulated reporter gene activity, and this stimulation was prevented with actinomycin D, indicating transcriptional activation. NaB effects on the NHE3 promoter depended on the activity of Ser/Thr kinases, in particular, protein kinase A (PKA). However, PKA stimulation alone did not have an effect on promoter activity, and it did not act synergistically with NaB. Another HDAC inhibitor, Trichostatin A (TSA), stimulated NHE3 promoter in a Ser/Thr kinase-independent fashion. The putative NaB-responsive elements were localized within −320/−34 bp of the NHE3 promoter. These findings suggest that PKA mediates NaB effects on NHE3 gene transcription and that the mechanism of NaB action is different from that of TSA.

Molecular mechanism of rat NHE3 gene promoter regulation by sodium butyrate

2007 ◽  
Vol 293 (1) ◽  
pp. C64-C74 ◽  
Author(s):  
Pawel R. Kiela ◽  
Nesrin Kuscuoglu ◽  
Anna J. Midura ◽  
Monica T. Midura-Kiela ◽  
Claire B. Larmonier ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Sodium Butyrate ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Upstream Site ◽  
Potent Inducer ◽  
Maximal Induction ◽  
Gel Mobility Shift

Sodium butyrate (NaB) stimulates sodium and water absorption by inducing colonic Na+/H+ exchange. NaB induces Na+/H+ exchanger (NHE)3 activity and protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is Ser/Thr kinase dependent and that NaB-responsive elements were localized within −320/−34 bp of the rat NHE3 promoter. Here we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient and stable transfection of Caco-2 cells with NHE3 gene reporter constructs identified Sp binding site SpB at position −58/−55 nt as critical for NaB-mediated induction. Gel mobility shift (GMSA) and DNA affinity precipitation assays indicated NaB-induced binding of Sp3 and decreased binding of Sp1 to SpB element. While no changes in expression of Sp1 or Sp3 were noted, NaB induced phosphorylation of Sp1 and acetylation of Sp3. Sp3 was a more potent inducer of NHE3 gene transcription, which suggested that change in balance, favoring binding of Sp3 to the SpB site, would result in significant increase in NHE3 promoter activity. Small interfering RNA studies in Caco-2 cells and data from NaB-treated SL2 cells used as a reconstitution model confirmed this hypothesis. In addition to the SpB site, which played a permissive role, an upstream novel butyrate response element located at −196/−175 nt was necessary for maximal induction. GMSA identified a protein-DNA complex with a −196/−175 nt probe; this interaction was not affected by NaB treatment, thus suggesting that in response to NaB Sp3 binding to site SpB precedes and results in recruitment of the putative factor to this upstream site.

scholarly journals Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6

1999 ◽  
Vol 276 (6) ◽  
pp. C1374-C1382 ◽  
Author(s):  
Susan A. Berry ◽  
Pearl L. Bergad ◽  
Allison M. Stolz ◽  
Howard C. Towle ◽  
Sarah Jane Schwarzenberg
Keyword(s):  
Gene Expression ◽  
Acute Phase ◽  
Gene Promoter ◽  
Rat Hepatocytes ◽  
Acute Phase Reactants ◽  
Primary Rat Hepatocytes ◽  
Fold Induction ◽  
Vitro Model

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a γ-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the −319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5.4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.

scholarly journals In vitro transcription from the human A gamma-globin gene promoter

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1344-1350 ◽  
Author(s):  
M Castle ◽  
D O'Neill ◽  
A Bank
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Systematic Analysis ◽  
Gamma Globin ◽  
Octamer Motif

We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

scholarly journals Spliced Leader RNA Gene Transcription in Trypanosoma brucei Requires Transcription Factor TFIIH

2007 ◽  
Vol 6 (4) ◽  
pp. 641-649 ◽  
Author(s):  
Ju Huck Lee ◽  
Tu N. Nguyen ◽  
Bernd Schimanski ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Gene Transcription ◽  
Transcription Initiation ◽  
Cell Cycle Control ◽  
Gene Promoter ◽  
Spliced Leader

ABSTRACTTrypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL)transsplicing and polyadenylation. Intranssplicing, the SL RNA is consumed through a transfer of its 5′-terminal part to the 5′ end of mRNAs. Since all mRNAs aretransspliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription inTrypanosoma brucei, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. AlthoughT. bruceiTFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether aT. bruceiTFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD inT. bruceiaffected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.

Q-mediated late gene transcription of bacteriophage λ: RNA start point and RNase III processing sites in vivo

Virology ◽  
1988 ◽  
Vol 167 (2) ◽  
pp. 568-577 ◽  
Author(s):  
D DANIELS ◽  
M SUBBARAO ◽  
F BLATTNER ◽  
H LOZERON
Keyword(s):  
Gene Transcription ◽  
Late Gene ◽  
Bacteriophage Λ ◽  
Rnase Iii
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