IMMUNOHISTOCHEMICAL DETECTION OF HUMAN CELLULAR YES GENE (c-yes-1) MESSENGER RNA BY A NON-RADIOACTIVE SYNTHESIZED OLIGODEOXYNUCLEOTIDE PROBE

抄録

A synthetic 39-mer anti-sense strand oligodeoxynucleotide complementary to the region of human cellular yes gene (c-yes-1) mRNA that codes for a polypeptide of 543 amino acids (Mr: 60, 801) and the corresponding sense strand 39-mer oligodeoxynucleotide were chemically synthesized by β-cyanoethyl phospho-ramidite method. The probes thymine-thymine (T-T) dimerized by UV irradiation were used for in situ hybridization studies. Various cells were fixed with ethanol-acetic acid (3:1) at room temperature for 20min and hybridization was carried out in 50% formamide for 36hr at 37°C. The good signal was obtained by utilizing anti-T-T dimer serum (IgG frafction), peroxidase-labeled goat anti-rabbit IgG antibody, and diaminobenzidine-4-HCl and H₂O₂ as substrates. The anti-sense strand synthetic probe hybridized with cellular RNA of cultured cells with c-yes-1 mRNA, while the sense strand synthetic probe did not. The c-yes-1 mRNA was clearly detected in the cytoplasm of c-yes-1 gene product-expressing cultured cells by in situ hybridization using a T-T dimerized synthetic probe. Synthetic T-T dimerized oligodeoxynucleotides appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.