Multiple bandshift assay: rapid identification and cloning of DNA fragments containing specific protein-binding sites

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

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Maximal Activity of an Erythroid-specific Enhancer Requires the Presence of Specific Protein Binding Sites in Linked Promoters

Journal of Biological Chemistry ◽

10.1074/jbc.273.22.13593 ◽

1998 ◽

Vol 273 (22) ◽

pp. 13593-13598 ◽

Cited By ~ 10

Author(s):

Persis J. Amrolia ◽

Wesley Gabbard ◽

John M. Cunningham ◽

Stephen M. Jane

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Maximal Activity ◽

Specific Protein ◽

Protein Binding Sites

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1099 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1099-1106 ◽

Cited By ~ 15

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.

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A Bayesian mixture model for chromatin interaction data

Statistical Applications in Genetics and Molecular Biology ◽

10.1515/sagmb-2014-0029 ◽

2015 ◽

Vol 14 (1) ◽

Cited By ~ 3

Author(s):

Liang Niu ◽

Shili Lin

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Chromatin Interaction ◽

Molecular Technique ◽

Type I ◽

Gene Promoters ◽

Dna Fragments ◽

Modeling Framework ◽

Protein Binding Sites ◽

Chromatin Interactions

AbstractChromatin interactions mediated by a particular protein are of interest for studying gene regulation, especially the regulation of genes that are associated with, or known to be causative of, a disease. A recent molecular technique, Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), that uses chromatin immunoprecipitation (ChIP) and high throughput paired-end sequencing, is able to detect such chromatin interactions genomewide. However, ChIA-PET may generate noise (i.e., pairings of DNA fragments by random chance) in addition to true signal (i.e., pairings of DNA fragments by interactions). In this paper, we propose MC_DIST based on a mixture modeling framework to identify true chromatin interactions from ChIA-PET count data (counts of DNA fragment pairs). The model is cast into a Bayesian framework to take into account the dependency among the data and the available information on protein binding sites and gene promoters to reduce false positives. A simulation study showed that MC_DIST outperforms the previously proposed hypergeometric model in terms of both power and type I error rate. A real data study showed that MC_DIST may identify potential chromatin interactions between protein binding sites and gene promoters that may be missed by the hypergeometric model. An R package implementing the MC_DIST model is available at

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