scholarly journals Analysis of transcription factors by gel shift and in-gel kinase assays-Gel shift assay.

1998 ◽  
Vol 42 (2) ◽  
pp. 93-96
Author(s):  
Takumi Kawabe ◽  
Toshifumi Tetsuka ◽  
Takashi Okamoto
Keyword(s):  
Transcription Factors ◽  
Gel Shift Assay ◽  
Gel Shift

scholarly journals Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Yong Feng ◽  
Yunfei Liao ◽  
Jianming Zhang ◽  
Jacson Shen ◽  
Zengwu Shao ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Transcriptional Analysis ◽  
Luciferase Assay ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Gel Shift Assay ◽  
Gel Shift

Abstract Background Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. Methods In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFβ) were further accessed in osteosarcoma. Results We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFβ expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. Conclusions These data establish CBFβ as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Graphical Abstract

scholarly journals Gel Shift Assay of Nuclear Extracts from Histoplasma capsulatum Demonstrates the Presence of Several DNA Binding Proteins

2002 ◽  
Vol 70 (4) ◽  
pp. 2238-2241 ◽  
Author(s):  
Atanas Ignatov ◽  
Elizabeth J. Keath
Keyword(s):  
Dna Binding ◽  
Fungal Pathogens ◽  
Binding Proteins ◽  
Histoplasma Capsulatum ◽  
Protein S ◽  
Dna Binding Proteins ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Shift Assay ◽  
Gel Shift

ABSTRACT A gel shift assay was optimized to detect several general DNA binding proteins from Histoplasma capsulatum strain G217B. The electrophoretic mobility shift assay (EMSA) technique also detected protein(s) recognizing a pyrimidine-rich motif found in several Histoplasma promoters. Establishment of EMSA conditions provides an important framework to evaluate regulation of homeostatic or phase-specific genes that may influence virulence in Histoplasma and other dimorphic fungal pathogens.

Detection of Heat Shock Element-Binding Activities by Gel Shift Assay During Mouse Preimplantation Development

1994 ◽  
Vol 165 (2) ◽  
pp. 627-638 ◽  
Author(s):  
Valérie Mezger ◽  
Jean-Paul Renard ◽  
Elisabeth Christians ◽  
Michel Morange
Keyword(s):  
Heat Shock ◽  
Preimplantation Development ◽  
Heat Shock Element ◽  
Gel Shift Assay ◽  
Gel Shift

scholarly journals Specific Contacts between Residues in the DNA-Binding Domain of the TyrR Protein and Bases in the Operator of thetyrP Gene of Escherichia coli

1999 ◽  
Vol 181 (8) ◽  
pp. 2338-2345 ◽  
Author(s):  
J. S. Hwang ◽  
J. Yang ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Binding Affinities ◽  
Wild Type ◽  
Small Reduction ◽  
Gel Shift Assay ◽  
Gel Shift

ABSTRACT In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051–1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the ±5 positions in the TyrR boxes.

scholarly journals A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene.

1990 ◽  
Vol 10 (8) ◽  
pp. 3884-3895 ◽  
Author(s):  
R M Luche ◽  
R Sumrada ◽  
T G Cooper
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Point Mutations ◽  
Saturation Mutagenesis ◽  
Gel Shift Assay ◽  
Dna Fragment ◽  
Gel Shift ◽  
Car1 Gene ◽  
Yeast Genes ◽  
Flanking Regions

Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS). Deletion of this element results in high-level expression of the CAR1 gene without inducer. To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis. Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3'. A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay. DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors. Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS. These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding. These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site. Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes. The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.

scholarly journals Role of Basic-Helix-Loop-Helix Transcription Factors in Sertoli Cell Differentiation: Identification of an E-Box Response Element in the Transferrin Promoter*

Endocrinology ◽  
1997 ◽  
Vol 138 (2) ◽  
pp. 667-675 ◽  
Author(s):  
Jaideep Chaudhary ◽  
Andrea S. Cupp ◽  
Michael K. Skinner
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Helix Loop Helix ◽  
Nuclear Extracts ◽  
E Box ◽  
Bhlh Factors ◽  
Gel Shift ◽  
Bhlh Proteins

Abstract Sertoli cells are critical for testicular function and maintenance of the spermatogenic process. The induction of Sertoli cell differentiation in the embryo promotes testicular development and male sex determination. The progression of Sertoli cell differentiation during puberty promotes the onset of spermatogenesis. The maintenance of optimal Sertoli cell differentiation in the adult is required for spermatogenesis to proceed. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation through the analysis of a previously identified marker of differentiation, transferrin gene expression. Sertoli cells produce transferrin to transport iron to developing spermatogenic cells sequestered within the blood-testis barrier. The transferrin promoter was characterized and found to contain two critical response elements, designated Sertoli element 1 (SE1) and Sertoli element 2 (SE2). Through sequence analysis, SE2 was found to contain an E-box response element, which has been shown to respond to basic-helix-loop-helix (bHLH) transcription factors. The bHLH proteins are a class of transcription factors associated with the induction and progression of cell differentiation. bHLH proteins dimerize through the conserved helix-loop-helix region and bind DNA through the basic region. Nuclear extracts from Sertoli cells were found to cause an E-box gel shift when the cells were stimulated to differentiate in culture, but not under basal conditions. The SE2 gel shift of Sertoli nuclear extracts was competed with excess unlabeled SE2 or E-box DNA fragments. Several Sertoli nuclear proteins associate with the SE2 gel shifts, including 70-, 42-, and 25-kDa proteins. Therefore, the critical SE2 element in the transferrin promoter is an E-box element capable of binding bHLH transcription factors. The ubiquitously expressed E12 bHLH protein dimerizes with numerous cell-specific bHLH factors. A Western blot analysis demonstrated that E12 was present in Sertoli cell nuclear extracts and associated with the SE2 gel shift. A ligand blot of Sertoli cell nuclear extracts with radiolabeled E12 had apparent bHLH proteins when the cells were stimulated to differentiate. The E-box sequence in the SE2 fragment of the transferrin promoter was CATCTG and was similar in gel shifts to the consensus E-box elements (CANNTG) previously characterized. A bHLH inhibitory factor (Id) competed and inhibited formation of the Sertoli cell nuclear extract E-box gel shift. To extend this observation, Id protein was overexpressed in cultured Sertoli cells. A transferrin promoter chloramphenicol acetyltransferase construct was used to monitor Sertoli cell function. The presence of Id suppressed the activation of the promoter induced by Sertoli differentiation factors. Therefore, the inhibition of Sertoli bHLH factors by Id suppressed Sertoli cell differentiated function, as measured by transferrin expression. An E-box-chloramphenicol acetyltransferase construct was also found to be active in Sertoli cells when cells were induced to differentiate. Screening the computerized nucleotide data bases demonstrated that putative E-box response elements are present in the promoters of a large number of Sertoli cell differentiated genes. In summary, a critical E-box response element has been identified in the transferrin promoter that can be activated by bHLH factors (e.g. E12) present in Sertoli cells. Inhibition of Sertoli bHLH factors by Id suppresses Sertoli cell differentiated function (i.e. transferrin expression), suggesting that bHLH transcription factors may be important in regulating Sertoli cell differentiated functions.

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