In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence.
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 it 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.
Harel-Bellan
A
A
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21701.
Brini
A T
AT
Ferris
D K
DK
Robin
P
P
Farrar
W L
WL
D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D006360:Heat-Shock Proteins / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015458:Leukemia, T-Cell / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured / Q000032:analysis
D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D015500:Chloramphenicol O-Acetyltransferase