EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

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.

scholarly journals Transcription signals and protein binding sites for sericin gene transcription in vitro

1989 ◽  
Vol 264 (31) ◽  
pp. 18707-18713 ◽  
Author(s):  
K Matsuno ◽  
C C Hui ◽  
S Takiya ◽  
T Suzuki ◽  
K Ueno ◽  
...  
Keyword(s):  
Protein Binding ◽  
Gene Transcription ◽  
Binding Sites ◽  
Protein Binding Sites ◽  
Transcription In Vitro ◽  
Transcription Signals

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

1998 ◽  
Vol 273 (22) ◽  
pp. 13593-13598 ◽  
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

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.

Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen

1994 ◽  
Vol 14 (1) ◽  
pp. 93-103
Author(s):  
P Jacquemin ◽  
C Oury ◽  
B Peers ◽  
A Morin ◽  
A Belayew ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Chromosome 17 ◽  
Placental Lactogen ◽  
Cell Nuclei ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Human Genes ◽  
Genes Encoding

The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.

scholarly journals Characterization of Specific Protein-RNA Complexes Associated with the Coupling of Polyadenylation and Last-Intron Removal

2002 ◽  
Vol 22 (13) ◽  
pp. 4579-4586 ◽  
Author(s):  
Charles Cooke ◽  
James C. Alwine
Keyword(s):  
Specific Protein ◽  
Polyadenylation Signal ◽  
Site Formation ◽  
Coupling Mechanism ◽  
Experimental Conditions ◽  
Specific Complex ◽  
Intron Removal ◽  
Definition Of ◽  
Rna Complexes

ABSTRACT Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (AC′, AC, and BC) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The AC′ complex is formed by 30 s after mixing, the AC complex is formed between 1 and 2 min after mixing, and the BC complex is formed by 2 to 3 min after mixing. AC′ is a precursor of AC, and the AC′ and/or AC complex is a precursor of BC. Of the three complexes, BC appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the AC′ complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the AC′ complex is dependent on the polypyrimidine tract. The transition from AC′ to AC appears to require an intact 3′-splice site. Formation of the BC complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the AC′ complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the BC complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.

An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro

2017 ◽  
Vol 59 (2-3) ◽  
pp. 59-65
Author(s):  
Liangyan Wang ◽  
Huizhi Lu ◽  
Yunguang Wang ◽  
Su Yang ◽  
Hong Xu ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Improved Method ◽  
Protein Binding Sites

scholarly journals Conserved Structure Modules within the lncRNA SChLAP1 Mediate Protein Recognition Implicated in Aggressive Prostate Cancer

2021 ◽  
Author(s):  
Emily J McFadden ◽  
James P Falese ◽  
Amanda E Hargrove
Keyword(s):  
Prostate Cancer ◽  
Secondary Structure ◽  
Protein Binding ◽  
Binding Sites ◽  
Cancer Metastasis ◽  
Magnesium Concentration ◽  
Purification Method ◽  
Aggressive Prostate Cancer ◽  
Protein Binding Sites

The lncRNA Second Chromosome Locus Associated with Prostate 1 (SChLAP1) was previously identified as a predictive biomarker and driver of aggressive prostate cancer. Recent work suggests that SChLAP1 may bind the SWI/SNF chromatin remodeling complex to promote prostate cancer metastasis, though the exact role of SWI/SNF recognition is debated. To date, there are no detailed biochemical studies of apo SChLAP1 or the SChLAP1:SWI/SNF complex. Herein, we report the first secondary structure model of SChLAP1 utilizing SHAPE-MaP both in vitro and in cellulo. Comparison of the in vitro and in cellulo data via ΔSHAPE identified putative protein binding sites within SChLAP1, specifically to evolutionarily conserved exons of the transcript. We also demonstrate that global SChLAP1 secondary structure is sensitive to both purification method and magnesium concentration. Further, we identified a 3'-fragment of SChLAP1 (SChLAP1Frag) that harbors multiple potential protein binding sites and presents a robustly folded secondary structure, supporting a functional role for this region. This work lays the foundation for future efforts in selective targeting and disruption of the SChLAP1:protein interface and the development of new therapeutic avenues in prostate cancer treatment.

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