In Situ Binding for Detection of Proteins That Bind to Regulatory Elements

Abstract

In situ binding assay with oligonucleotides is developed for detection of DNA-binding factors. Cryosections of porcine pituitary were examined by DNA binding with 35S-labeled oligonucleotides corresponding to the consensus cAMP-responsive element (CRE) and the activator protein-1 binding element (AP1). Specificity of the in situ binding was confirmed by inhibition with the unlabeled probe itself and inefficiency of binding with the activator protein-2 binding element (AP2). It was proved by treatment with proteinase K that the binding was dependent on the protein component. Immunocytochemical staining employed successively before in situ binding demonstrated that the binding proteins for CRE and AP1 are located in pituitary gonadotrophs as well as other cells. Grain counting showed that gonadotrophs were rich in these factors as compared with other pituitary cells. Taken together, in situ binding provides a convenient and rapid tool for investigation of regulatory sequences and binding factors as well for determining their specific cellular localization.