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Complex formation with the fibroin gene enhancer through a protein-protein interaction analyzed by a modified DNA-binding assay.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1989 Mar 15; Vol. 264 (8), pp. 4599-604. - Publication Year :
- 1989
-
Abstract
- The distal upstream region of the fibroin gene, -238 to -73, is known to contain the cis-acting element(s) responsible for tissue-specific transcription enhancement in vitro. To identify factor(s) trans-acting on this region, we have developed a modified nitrocellulose blot protein-DNA binding procedure that detects DNA-protein complexes via protein-protein interaction. A protein that does not bind to DNA as a monomer cannot be detected by standard Southwestern blotting. To detect protein-DNA complexes involving both protein-DNA and protein-protein interactions, the protein blot was reacted with the extract protein to form a protein complex on the filter, in the presence of the DNA probe. This new method revealed that the 75-kDa protein binds to the enhancer region of the fibroin gene in the presence of the second protein(s). The 75-kDa protein and the factor(s) mediating DNA binding activity were recovered in two different column fractions. The level of the 75-kDa protein appeared to be much higher in silk gland extracts than in nonsilk gland extracts, whereas the mediating factor(s) distributed evenly. These observations suggest that the distal upstream region of the fibroin gene can be specifically recognized by a protein complex composed of at least two distinct proteins, through protein-protein interaction.
- Subjects :
- Animals
Bombyx analysis
DNA Probes
Electrophoresis, Polyacrylamide Gel
Exocrine Glands analysis
Hot Temperature
Molecular Weight
Nucleic Acid Hybridization
Tissue Distribution
Trypsin pharmacology
Bombyx genetics
DNA metabolism
Enhancer Elements, Genetic
Fibroins genetics
Transcription Factors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 264
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 2925657