DNase I Footprinting

2009 ◽  
pp. 153-172 ◽  
Author(s):  
Antonia S. Cardew ◽  
Keith R. Fox
Keyword(s):  
Dnase I ◽  
Dnase I Footprinting

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

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.

Genomic footprinting detects factors bound to major late and IVa2 promoters in adenovirus-infected HeLa cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1534-1539
Author(s):  
G Albrecht ◽  
B Devaux ◽  
C Kedinger
Keyword(s):  
Protein Interactions ◽  
Adenovirus Type ◽  
Nuclease Activity ◽  
Dnase I ◽  
Promoter Activation ◽  
Promoter Elements ◽  
Dnase I Footprinting ◽  
Infected Cells ◽  
Adenovirus Type 5

We used DNase I footprinting assays on nuclei isolated from adenovirus-infected cells to examine the nucleoprotein configuration of a 250-base-pair segment which encompasses the adenovirus type 5 major late (ML) and IVa2 promoters. At 12 and 20 h postinfection (p.i.), fine DNase I digestion mapping of wild-type adenovirus-infected cells revealed specific sequences protected from digestion which corresponded to promoter elements required for expression of the ML gene in vivo. At 12 h p.i., a G+C-rich region which lies upstream of the IVa2 cap site and is important for maximal IVa2 activity was also found masked to nuclease activity. At 20 h p.i., however, this element became more sensitive to nuclease attack, while the ML promoter elements stayed protected. No major changes in DNA-protein interactions were detected in the region spanning the ML and IVa2 cap sites upon promoter activation, suggesting that the binding properties of the cognate factors for this region are not modified during the process.

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 Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes inStreptomyces coelicolor

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Meng Liu ◽  
Peipei Zhang ◽  
Yanping Zhu ◽  
Ting Lu ◽  
Yemin Wang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Consensus Sequence ◽  
Aerial Mycelium ◽  
Dnase I ◽  
Inverted Repeats ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Two Component

ABSTRACTAs with most annotated two-component systems (TCSs) ofStreptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, formorphogenesis andactinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using anS. coelicolorstrain expressing MacR-Flag fusion protein, identifiedin vivotargets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assaysin vitroand by ChIP-quantitative PCRin vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes,sco6728,sco4924, andsco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS fromS. avermitilisandS. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism inStreptomyces.IMPORTANCETCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in theStreptomycesmodel strainS. coelicolorare unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that otherStreptomycesspecies have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems inStreptomyces.

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 HpdR Is a Transcriptional Activator of Sinorhizobium meliloti hpdA, Which Encodes a Herbicide-Targeted 4-Hydroxyphenylpyruvate Dioxygenase

2007 ◽  
Vol 189 (9) ◽  
pp. 3660-3664 ◽  
Author(s):  
Suvit Loprasert ◽  
Wirongrong Whangsuk ◽  
James M. Dubbs ◽  
Ratiboot Sallabhan ◽  
Kumpanart Somsongkul ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Transcriptional Activator ◽  
Direct Repeat ◽  
Dnase I ◽  
Mobility Shift ◽  
Intergenic Space ◽  
Repeat Sequences ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

scholarly journals Dissociation of the AT-specific bifunctional intercalator [N-MeCys3,N-MeCys7]TANDEM from TpA sites in DNA

1995 ◽  
Vol 306 (1) ◽  
pp. 15-19 ◽  
Author(s):  
M C Fletcher ◽  
R K Olsen ◽  
K R Fox
Keyword(s):  
Dissociation Rate ◽  
Dnase I ◽  
Time Dependent ◽  
Base Pairs ◽  
Unusual Structure ◽  
Dnase I Footprinting ◽  
Calf Thymus ◽  
Stability Of Complexes ◽  
Dna Fragment ◽  
The Stability

We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.

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