17 Introduction to the Detection of Transcription Regulatory Proteins
241
to find the report in which pBR 322 DNA contains glucocorticoid
responsive element (GRE) consensus sequences at five sites and
actually can bind to GR on southwestern blots (Tully and Cidlowski 1987). Our previous finding now can be interpreted in a
way that pBR 322 DNA may bind to GR in the tissue section. This
interpretation is also consistent with the fact that GR consists of
three autonomous domains; trans-activation domain, DNAbinding domain and hormone-binding domain, and regulates
gene expression through its binding to specific DNA sequences,
called "GRE", located in the vicinity of the regulated gene (Evans
1988; Beato 1989).
Based upon the accumulated data, we attempted to localize
GR by utilizing the specific interaction between GR and GRE (later the method was named Southwestern histochemistry (Koji et
al 1994) owing to the apparent similarity in principle to that of
southwestern analysis (Miskimins et al. 1985)); frozen sections of
rat liver were reacted with thymidine-thymidine (T -T) dimerized
pBR 322 DNA and then the T -T dimers were detected enzymeimmunohistochemically by the successive use of rabbit anti- (TT dimer) antibody and HRP-anti-(rabbit IgG). Consequently,
specific staining was confirmed in the nuclei, nuclear membranes as well as cytoplasm of hepatocytes (Koji et al. 1990,
1992). The success encouraged us to combine the method
with the use of synthetic double-stranded oligo-DNA, as described schematically in Fig. 3. In principle, the use of haptenic
oligo-DNA is more specific and more convenient than segments
of DNA (Koji et al. 1992) or radiolabeled oligo-DNA.
As a model system to demonstrate the usefulness of haptenized oligo-DNA for the localization of specific transcription factors at the level of individual cells, cyclic adenosine 3',5' -monophosphate (cAMP) responsive element (CRE) binding protein
was localized in the frozen sections of rat brain and small intestine (Fig. 4) (Koji et al. 1994). We synthesized the CRE consensus
base sequence (TGACGTCA) together with three TTA repeats at
5' -end and allowed it to be annealed, as given below:
** ** **
ITA ITA IT A-TGACGTCA
ACTGCAGT-ATTAITAIT
** ** **
(**) = possible sites ofT-T dimers
241
to find the report in which pBR 322 DNA contains glucocorticoid
responsive element (GRE) consensus sequences at five sites and
actually can bind to GR on southwestern blots (Tully and Cidlowski 1987). Our previous finding now can be interpreted in a
way that pBR 322 DNA may bind to GR in the tissue section. This
interpretation is also consistent with the fact that GR consists of
three autonomous domains; trans-activation domain, DNAbinding domain and hormone-binding domain, and regulates
gene expression through its binding to specific DNA sequences,
called "GRE", located in the vicinity of the regulated gene (Evans
1988; Beato 1989).
Based upon the accumulated data, we attempted to localize
GR by utilizing the specific interaction between GR and GRE (later the method was named Southwestern histochemistry (Koji et
al 1994) owing to the apparent similarity in principle to that of
southwestern analysis (Miskimins et al. 1985)); frozen sections of
rat liver were reacted with thymidine-thymidine (T -T) dimerized
pBR 322 DNA and then the T -T dimers were detected enzymeimmunohistochemically by the successive use of rabbit anti- (TT dimer) antibody and HRP-anti-(rabbit IgG). Consequently,
specific staining was confirmed in the nuclei, nuclear membranes as well as cytoplasm of hepatocytes (Koji et al. 1990,
1992). The success encouraged us to combine the method
with the use of synthetic double-stranded oligo-DNA, as described schematically in Fig. 3. In principle, the use of haptenic
oligo-DNA is more specific and more convenient than segments
of DNA (Koji et al. 1992) or radiolabeled oligo-DNA.
As a model system to demonstrate the usefulness of haptenized oligo-DNA for the localization of specific transcription factors at the level of individual cells, cyclic adenosine 3',5' -monophosphate (cAMP) responsive element (CRE) binding protein
was localized in the frozen sections of rat brain and small intestine (Fig. 4) (Koji et al. 1994). We synthesized the CRE consensus
base sequence (TGACGTCA) together with three TTA repeats at
5' -end and allowed it to be annealed, as given below:
** ** **
ITA ITA IT A-TGACGTCA
ACTGCAGT-ATTAITAIT
** ** **
(**) = possible sites ofT-T dimers