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Localization of cyclic adenosine 3',5'-monophosphate-responsive element (CRE)-binding proteins by southwestern histochemistry
T Koji, K Komuta, M Nozawa, S Yamada and PK Nakane
Department of Anatomy, Nagasaki University School of Medicine, Japan.
Regulation of gene transcription requires an interaction between specific segments of nuclear DNA and specific proteins. We describe a method to localize the specific DNA-binding proteins using haptenized double-stranded (ds) DNAs. To demonstrate this method, an oligodeoxynucleotide (oligo-DNA) with a consensus base sequence of cyclic adenosine monophosphate-responsive element (CRE) (TGACGTCA) with three TTA repeats at the 5' end was synthesized. Since the CRE sequence is palindromic, the oligo-DNA was allowed to self-anneal and form ds DNA with three TTA repeats at both ends. The CRE ds-oligo-DNA was irradiated with UV light to form haptenic thymine-thymine (T-T) dimers. The haptenized CRE ds-oligo-DNA reacted by Southwestern analysis with a distinct set of proteins, previously identified as CRE-binding proteins, ranging from 40-90 KD. When the haptenized CRE ds-oligo-DNA reacted with frozen sections fixed with 4% paraformaldehyde in PBS (pH 7.4) followed by enzyme immunohistochemical localization of the T-T dimers, nuclei of intestinal epithelial cells and brain cells were heavily stained. Nuclear staining was blocked when the sections were reacted with the haptenized CRE ds-oligo-DNA in the presence of an excess amount of non-haptenized CRE ds-oligo-DNA. This method, henceforth referred as Southwestern histochemistry, should be a useful tool to localize proteins that bind to a specific DNA sequence and regulate the transcriptional activity of genes.
Volume 42,
Issue 10,
pp. 1399-1405,
10/01/1994
Copyright © 1994 by The Histochemical Society
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