Localization of cyclic adenosine 3',5'-monophosphate-responsive element (CRE)-binding proteins by southwestern histochemistry.
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.
Koji
T
T
Department of Anatomy, Nagasaki University School of Medicine, Japan.
Komuta
K
K
Nozawa
M
M
Yamada
S
S
Nakane
P K
PK
D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D000242:Cyclic AMP / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D006651:Histocytochemistry / Q000379:methods*; D015151:Immunoblotting / Q000379:methods; D007421:Intestine, Small / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D051381:Rats; D017208:Rats, Wistar; D012045:Regulatory Sequences, Nucleic Acid
D015342:DNA Probes; D004268:DNA-Binding Proteins; D000242:Cyclic AMP