The Proteolytic Clipping Band-Shift Assay of Protein-DNA Complexes

1991 ◽  
pp. 45-54
Author(s):  
Melya J. Hughes ◽  
Haimin Liang ◽  
Jean-Pierre Jost
Keyword(s):  
Dna Complexes ◽  
Band Shift ◽  
Band Shift Assay

The Xenopus B1 factor is closely related to the mammalian activator USF and is implicated in the developmental regulation of TFIIIA gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 412-424
Author(s):  
H Kaulen ◽  
P Pognonec ◽  
P D Gregor ◽  
R G Roeder
Keyword(s):  
Developmental Regulation ◽  
Human Adenovirus ◽  
Regulatory Proteins ◽  
Cdna Libraries ◽  
Promoter Activation ◽  
Dna Complexes ◽  
Band Shift ◽  
Sequence Identity ◽  
Helix Loop Helix ◽  
Late Transcription

The Xenopus laevis TFIIIA promoter contains a motif that has been implicated in promoter activation in late-stage oocytes and contains the sequence (-269) CACGTG (-264). A cDNA encoding a protein (B1) that binds to this element has been cloned from X. laevis and Xenopus borealis ovarian cDNA libraries. We show that this protein is a member of the helix-loop-helix family of regulatory proteins and contains 80% sequence identity with the human adenovirus major late transcription factor (MLTF or USF). A survey of B1 protein expression during oogenesis and embryogenesis revealed both oocyte-specific and somatic cell-specific B1 protein-DNA complexes. Immunological data, RNA blot analysis, and proteolytic clipping band shift assays indicated that these complexes most likely represent altered forms of a single B1 polypeptide. Implications for TFIIIA gene regulation during development are discussed.

scholarly journals Protein-DNA interactions associated with the onset of testis-specific expression of the mammalian Pgk-2 gene.

1992 ◽  
Vol 12 (4) ◽  
pp. 1422-1431 ◽  
Author(s):  
M M Gebara ◽  
J R McCarrey
Keyword(s):  
Dna Binding ◽  
Nuclear Protein ◽  
Core Promoter ◽  
Somatic Cells ◽  
Dna Interactions ◽  
Band Shift ◽  
Specific Expression ◽  
Protein Dna Interactions ◽  
Gc Box ◽  
Band Shift Assay

We have identified difference in protein-DNA interactions associated with the promoter of the mammalian spermatogenesis-specific Pgk-2 gene in expressing and nonexpressing cells, using a band shift assay. We compared DNA-binding activities in nuclear protein extracts from expressing adult testis cells versus nonexpressing prepuberal testis cells and nonexpressing somatic cells. One or two DNA-binding activities were found to be uniquely associated with the expressed state of Pgk-2, while a third appears to be associated with the nonexpressed state. All three of these activities map to a region within the first 40 bp upstream from the core promoter of this gene. The Pgk-2 core promoter lacks a TATA box but contain a GC box and a CAAT box. We show that the GC box binds the ubiquitous transcription factor Sp1 and that the CAAT box binds CTF-1, both of which are present in extracts from all three tissue types tested. These results suggest that tissue-specific transcription of the Pgk-2 gene is associated with changes in protein-DNA interactions occurring within a 40-bp enhancer region and that different arrays of protein-DNA interactions in this region are associated with the actively expressed state of the Pgk-2 gene in spermatocytes and spermatids and with the terminally repressed state of Pgk-2 in somatic cells.

scholarly journals NFAT but not NF-κB is critical for transcriptional induction of the prosurvival gene A1 after IgE receptor activation in mast cells

Blood ◽  
2008 ◽  
Vol 111 (6) ◽  
pp. 3081-3089 ◽  
Author(s):  
Erik Ullerås ◽  
Mats Karlberg ◽  
Christine Möller Westerberg ◽  
Jessica Alfredsson ◽  
Steve Gerondakis ◽  
...  
Keyword(s):  
Mast Cells ◽  
Cyclosporin A ◽  
Receptor Activation ◽  
Luciferase Expression ◽  
Band Shift ◽  
Monocytic Cells ◽  
Inducible Protein ◽  
And Function ◽  
Transcriptional Induction ◽  
Band Shift Assay

Abstract FcϵRI-activation–induced survival of mast cells is dependent on the expression and function of the prosurvival protein A1. The expression of A1 in lymphocytes and monocytes has previously been described to be transcriptionally regulated by NF-κB. Here we demonstrate that the expression of A1 in mast cells is not dependent on NF-κB but that NFAT plays a crucial role. FcϵRI-induced A1 expression was not affected in mast cells overexpressing an IκB-α super-repressor or cells lacking NF-κB subunits RelA, c-Rel, or c-Rel plus NF-κB1 p50. In contrast, inhibition of calcineurin and NFAT by cyclosporin A abrogated the expression of A1 in mast cells on FcϵRI-activation but had no effect on lipopolysaccharide-induced expression of A1 in J774A.1 monocytic cells. Cyclosporin A also inhibited luciferase expression in an A1 promoter reporter assay. A putative NFAT binding site in the A1 promoter showed inducible protein binding after FcϵRI crosslinking or treatment with ionomycin as detected in a band shift assay or chromatin immunoprecipitation. The binding protein was identified as NFAT1. Finally, mast cells expressing constitutively active NFAT1 exhibit increased expression of A1 after FcϵRI-stimulation. These results indicate that, in FcϵRI stimulated mast cells, A1 is transcriptionally regulated by NFAT1 but not by NF-κB.

scholarly journals The abundance and in vitro DNA binding of three cellular proteins interacting with the adenovirus EIIa early promoter are not modified by the EIa gene products.

1987 ◽  
Vol 7 (10) ◽  
pp. 3806-3817 ◽  
Author(s):  
P Jalinot ◽  
B Devaux ◽  
C Kédinger
Keyword(s):  
Binding Sites ◽  
Adenovirus Type ◽  
Specific Protein ◽  
Gene Products ◽  
Wild Type ◽  
Band Shift ◽  
Transcription Complexes ◽  
Adenovirus Type 5 ◽  
Band Shift Assay

Specific protein binding on the EIa-inducible adenovirus EIIa early (EIIaE) promoter was analyzed by the sensitive electrophoretic band-shift assay and by protection against DNase I digestion. Three factors were identified, and precise mapping of the cognate-binding sites revealed their correspondence to promoter elements essential for constitutive EIIaE transcription. One binds to the major upstream element located between -82 and -64 (with respect to the major EIIaE cap site), another appears to interact with sequences on either side of this region, and the last one binds to an element located further upstream. Comparison of the binding activities of the factors present in extracts from cells infected with wild-type adenovirus (adenovirus type 5) or with the EIa deletion mutant dl312 did not reveal striking differences. Not only were the general binding patterns indistinguishable, but the concentration of each of the identified factors as well as their affinity for the cognate-binding sites were unchanged. Our results suggest that the EIa-mediated activation of the EIIaE transcription complexes involves appropriate interactions between transcription factors, rather than their increased binding to DNA.

scholarly journals Specific binding of estrogen receptor to the estrogen response element.

1989 ◽  
Vol 9 (1) ◽  
pp. 43-49 ◽  
Author(s):  
L Klein-Hitpass ◽  
S Y Tsai ◽  
G L Greene ◽  
J H Clark ◽  
M J Tsai ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Specific Binding ◽  
Rotational Symmetry ◽  
Binding Interaction ◽  
Band Shift ◽  
Estrogen Regulation ◽  
Contact Sites ◽  
Estrogen Response ◽  
Estrogen Response Elements ◽  
Band Shift Assay

Gene transfer studies have shown that estrogen regulation of specific genes is mediated by estrogen response elements (ERE). We report that binding of the estrogen receptor to the ERE can be detected by a gel retardation (band shift) assay. This binding interaction was highly sequence and receptor specific. Methylation interference analysis showed that the ERE contact sites of estrogen receptor displayed a perfect twofold rotational symmetry. This is compatible with estrogen receptor binding to the ERE as a head-to-head dimer.

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