Characterization of a new monoclonal antibody to triplex DNA and immunofluorescent staining of mammalian chromosomes.

A monoclonal antibody, Jel 466, was prepared from mice immunized with poly[d(Tm5C)].poly[d(GA)]. The binding of Jel 466 to nucleic acids was characterized by solid phase radioimmunoassays and competition experiments. There was no binding to single-stranded DNAs or to duplexes which could not form triplexes. In addition, the antibody preferred the triplex form of poly[d(TC)].poly[d(GA)]; it bound weakly to the triplex derived from poly[d(G)].poly[d(C)], but there was no interaction with poly[d(T)].poly[d(A)].poly[d(T)]. This pattern of specificity is very different from that of Jel 318, a triplex-specific antibody that will bind to poly[d(T)].poly[d(A)].poly[d(T)]. The amino acid sequence of Jel 466 also showed very little homology with Jel 318, although both contain many positively charged amino acids. The immunofluorescent staining of mouse and human chromosomes with Jel 466 was studied. In all cases, there was a marked reciprocal relationship between the pattern of Jel 466 on the one hand and that of Hoechst 33258 and Jel 318 on the other. Jel 466 was negative for C-band and G-band but positive for R-band, whereas the opposite was found for Hoechst and Jel 318. Since C and G-bands are AT-rich and R-bands are GC-rich, these staining patterns match the sequence preferences of the two antibodies. Thus the base composition of triplex-forming DNA differs from domain to domain.