Cloning and characterization of the 5′-flanking region of the rat glutamate-cysteine ligase catalytic subunit

2001 ◽  
Vol 357 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Heping YANG ◽  
Jiaohong WANG ◽  
Zong-Zhi HUANG ◽  
Xiaopeng OU ◽  
Shelly C. LU
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Start Site ◽  
Glutamate Cysteine Ligase ◽  
Dnase I Footprinting ◽  
H4iie Cells ◽  
Flanking Region

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis, is made up of two subunits, a catalytic (heavy) subunit (GCLC) and a modifier (light) subunit (GCLM), which are differentially regulated. Increased hepatic GCLC expression occurs during rapid growth, oxidative stress and after ethanol treatment. To facilitate studies of GCLC transcriptional regulation, we have cloned and characterized a 1.8kb 5′-flanking region of the rat GCLC (GenBank accession number AF218362). A consensus TATA box and one transcriptional start site are located at 302 and 197 nucleotides upstream of the translational start site, respectively. The promoter contains consensus binding sites for many transcription factors including nuclear factor κB (NF-κB) and activator protein 1 (AP-1). The rat GCLC promoter was able to efficiently drive luciferase expression in H4IIE cells. Sequential deletion analysis revealed that three DNA regions, −595 to −111, −1108 to −705 and −705 to −595, are involved in positive (the first two regions) and negative (the latter region) gene regulation. Specific protein binding to these regions was confirmed by DNase I footprinting and electrophoretic mobility-shift assays (EMSAs). Ethanol-fed livers exhibit increased protein binding to region −416 to −336 on DNase I footprinting analysis, which was found to be NF-κB and AP-1 on EMSA and supershift analysis. Acetaldehyde treatment of H4IIE cells led to a time- and dose-dependent increase in GCLC mRNA levels, binding of NF-κB and AP-1 to the GCLC promoter, and luciferase activity driven by the GCLC promoter fragment containing these binding sites.

scholarly journals Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

1987 ◽  
Vol 7 (6) ◽  
pp. 2059-2069 ◽  
Author(s):  
B Kemper ◽  
P D Jackson ◽  
G Felsenfeld
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Dnase I ◽  
Specific Factor ◽  
Mobility Shift ◽  
Protein Binding Sites ◽  
Dnase I Footprinting ◽  
Flanking Region

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene

1987 ◽  
Vol 7 (6) ◽  
pp. 2059-2069
Author(s):  
B Kemper ◽  
P D Jackson ◽  
G Felsenfeld
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Dnase I ◽  
Specific Factor ◽  
Mobility Shift ◽  
Protein Binding Sites ◽  
Dnase I Footprinting ◽  
Flanking Region

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

DNase I Footprinting to Identify Protein Binding Sites

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (14) ◽  
Author(s):  
Isabelle Gaugué ◽  
Dominique Bréchemier-Baey ◽  
Jacqueline Plumbridge
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Protein Binding Sites ◽  
Dnase I Footprinting

Mapping Protein-binding Sites on DNA by DNase I Footprinting

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot3947
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Protein Binding Sites ◽  
Dnase I Footprinting

scholarly journals Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1868-1874 ◽  
Author(s):  
HI Gomolin ◽  
Y Yamaguchi ◽  
AV Paulpillai ◽  
LA Dvorak ◽  
SJ Ackerman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Start Site ◽  
Human Eosinophil

Abstract The Charcot-Leyden crystal (CLC) protein is a lysophospholipase expressed exclusively by eosinophils and basophils. During eosinophilic differentiation of eosinophil-committed cell lines, CLC steady state mRNA levels increase significantly. This increased expression is transcriptionally regulated during butyrate induction of an eosinophilic subline (C15) of the promyelocytic leukemia cell line HL- 60, as shown by nuclear run-on assays. The transcriptional start site of the CLC gene was identified 43 bp upstream of the 5′ end of the longest available cDNA sequence. The gene encoding CLC protein was cloned from a chromosome 19-specific library and a fragment overlapping the transcriptional start site was isolated and sequenced. Plasmid constructs (in the pXP2 luciferase expression vector) containing 411 and 292 bp of genomic sequence upstream of the CLC transcriptional start site directed reporter gene expression in transient transfections of HL-60-C15 cells, as well as other myeloid (U937) and nonmyeloid (HeLa and RPMI 8402) cell lines. However, the differential expression of the two CLC promoter constructs in these cell lines suggests that the -292 to -411 bp region of the promoter may confer some specificity for expression in the eosinophil lineage. The CLC promoter sequence contains two consensus GATA binding sites, a purine-rich sequence that presents potential binding sites for PU.1, a member of the ets family of genes, as well as sequences described in other myeloid-specific promoters. This is the first demonstration of a functional eosinophil promoter that could serve as a model for identifying DNA elements and trans-activating factors that regulate gene expression during the commitment and differentiation of the eosinophil lineage.

scholarly journals Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1868-1874 ◽  
Author(s):  
HI Gomolin ◽  
Y Yamaguchi ◽  
AV Paulpillai ◽  
LA Dvorak ◽  
SJ Ackerman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Start Site ◽  
Human Eosinophil

The Charcot-Leyden crystal (CLC) protein is a lysophospholipase expressed exclusively by eosinophils and basophils. During eosinophilic differentiation of eosinophil-committed cell lines, CLC steady state mRNA levels increase significantly. This increased expression is transcriptionally regulated during butyrate induction of an eosinophilic subline (C15) of the promyelocytic leukemia cell line HL- 60, as shown by nuclear run-on assays. The transcriptional start site of the CLC gene was identified 43 bp upstream of the 5′ end of the longest available cDNA sequence. The gene encoding CLC protein was cloned from a chromosome 19-specific library and a fragment overlapping the transcriptional start site was isolated and sequenced. Plasmid constructs (in the pXP2 luciferase expression vector) containing 411 and 292 bp of genomic sequence upstream of the CLC transcriptional start site directed reporter gene expression in transient transfections of HL-60-C15 cells, as well as other myeloid (U937) and nonmyeloid (HeLa and RPMI 8402) cell lines. However, the differential expression of the two CLC promoter constructs in these cell lines suggests that the -292 to -411 bp region of the promoter may confer some specificity for expression in the eosinophil lineage. The CLC promoter sequence contains two consensus GATA binding sites, a purine-rich sequence that presents potential binding sites for PU.1, a member of the ets family of genes, as well as sequences described in other myeloid-specific promoters. This is the first demonstration of a functional eosinophil promoter that could serve as a model for identifying DNA elements and trans-activating factors that regulate gene expression during the commitment and differentiation of the eosinophil lineage.

Identical genomic footprints of the adenovirus EIIa promoter are detected before and after EIa induction

1987 ◽  
Vol 7 (12) ◽  
pp. 4560-4563
Author(s):  
B Devaux ◽  
G Albrecht ◽  
C Kedinger
Keyword(s):  
Transcription Factors ◽  
Protein Binding ◽  
Promoter Region ◽  
Adenovirus Type ◽  
Specific Protein ◽  
Dnase I ◽  
Dnase I Footprinting ◽  
Before And After ◽  
Adenovirus Type 5 ◽  
Transcriptional Induction

Genomic DNase I footprinting was used to compare specific protein binding to the adenovirus type 5 early, EIa-inducible, EIIa promoter. Identical protection patterns of the promoter region were observed whether EIIa transcription was undetectable or fully induced. These results suggest that EIa-mediated transcriptional induction does not increase binding of limiting transcription factors to the promoter but rather that transactivation results from the proper interactions between factors already bound to their cognate sequences.

scholarly journals CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5060-5067 ◽  
Author(s):  
M. Carolina Pilonieta ◽  
Maria D. Bodero ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Dnase I ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Dnase I Footprinting ◽  
Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

scholarly journals Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

1991 ◽  
Vol 11 (2) ◽  
pp. 1099-1106 ◽  
Author(s):  
F P Lemaigre ◽  
S M Durviaux ◽  
G G Rousseau
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Specific Protein ◽  
Regulatory Sequences ◽  
Alternative Promoters ◽  
Gene Encoding ◽  
Protein Binding Sites ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

scholarly journals Interaction of bleomycin with a bent DNA fragment

1992 ◽  
Vol 284 (3) ◽  
pp. 929-934 ◽  
Author(s):  
K P Nightingale ◽  
K R Fox
Keyword(s):  
Binding Sites ◽  
Dna Bending ◽  
Cobalt Complex ◽  
Dnase I ◽  
Cleavage Sites ◽  
Kinetoplast Dna ◽  
Bent Dna ◽  
Diethyl Pyrocarbonate ◽  
Dnase I Footprinting ◽  
Dna Fragment

The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3′ side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.

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