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In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence

Abstract

In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.
Authors:
Harel-Bellan, A; Brini, A T; Farrar, W L; [1]  Ferris, D K; [2]  Robin, P [3] 
  1. National Cancer Institute, Frederick, MD (USA)
  2. Program Resources, Inc., Frederick, MD (USA)
  3. Institut Gustave Roussy, Villejuif (France)
Publication Date:
Jun 12, 1989
Product Type:
Journal Article
Reference Number:
EDB-90-056272
Resource Relation:
Journal Name: Nucleic Acids Research; (UK); Journal Volume: 17:11
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; 59 BASIC BIOLOGICAL SCIENCES; MEMBRANE PROTEINS; CROSS-LINKING; OLIGONUCLEOTIDES; DNA SEQUENCING; AUTORADIOGRAPHY; GROWTH FACTORS; IN VITRO; MAN; MOLECULAR WEIGHT; PHOSPHORUS 32; PLASMIDS; TEMPERATURE DEPENDENCE; TUMOR CELLS; ULTRAVIOLET RADIATION; ANIMAL CELLS; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CELL CONSTITUENTS; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; ELECTROMAGNETIC RADIATION; ISOTOPES; LIGHT NUCLEI; MAMMALS; MITOGENS; NUCLEI; NUCLEIC ACIDS; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PHOSPHORUS ISOTOPES; POLYMERIZATION; PRIMATES; PROTEINS; RADIATIONS; RADIOISOTOPES; STRUCTURAL CHEMICAL ANALYSIS; VERTEBRATES; 550602* - Medicine- External Radiation in Diagnostics- (1980-); 550201 - Biochemistry- Tracer Techniques
OSTI ID:
7105347
Country of Origin:
United Kingdom
Language:
English
Other Identifying Numbers:
Journal ID: ISSN 0305-1048; CODEN: NARHA
Submitting Site:
JMT
Size:
Pages: 4077-4087
Announcement Date:

Citation Formats

Harel-Bellan, A, Brini, A T, Farrar, W L, Ferris, D K, and Robin, P. In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence. United Kingdom: N. p., 1989. Web. doi:10.1093/nar/17.11.4077.
Harel-Bellan, A, Brini, A T, Farrar, W L, Ferris, D K, & Robin, P. In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence. United Kingdom. doi:10.1093/nar/17.11.4077.
Harel-Bellan, A, Brini, A T, Farrar, W L, Ferris, D K, and Robin, P. 1989. "In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence." United Kingdom. doi:10.1093/nar/17.11.4077. https://www.osti.gov/servlets/purl/10.1093/nar/17.11.4077.
@misc{etde_7105347,
title = {In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence}
author = {Harel-Bellan, A, Brini, A T, Farrar, W L, Ferris, D K, and Robin, P}
abstractNote = {In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.}
doi = {10.1093/nar/17.11.4077}
journal = {Nucleic Acids Research; (UK)}
volume = {17:11}
journal type = {AC}
place = {United Kingdom}
year = {1989}
month = {Jun}
}