Nucleic Acids Research, 1989, Vol. 17, No. 11 4077-4087
© 1989
MOLECULAR BIOLOGY |
In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence
1Biological Carcinogenesis Development Program, Program Resources, Inc. Frederick Cancer Research Facility Frederick, MD 21701, USA 2Laboratoire d'Immunologie, Institut Gustave Roussy 39 rue Camille Desmoulins, 94805 Villejuif, France Laboratory of Molecular Immunoregulation, Biological Response Modifier Program Building 567, National Cancer Institute, Frederick Cancer Research Facility Frederick, MD 21701
Received March 1, 1989. Revised May 3, 1989. Accepted May 3, 1989.
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.
*Present address: Laboratoire d'Immunologie, CNRS UA 1156, Institut Gustave Roussy, Pavilion de Recherche 1, 39 rue Camille Desmoulins, 94805 Villejuif, France