Molecular Mechanics Studies of Homopolymeric and Mixed Sequence Py.Pu*Pu Triple Helices,

DNA triple helices containing two purine strands and one pyrimidine strand (C.G*G and T.A*A) have been studied, using model building followed by energy minimisation, for different orientations of the third strand resulting from variation in the hydrogen bonding between the Watson-Crick duplex and the third strand and the glycosidic torsion angle in the third strand. Our results show that in the C.G*G case the structure with a parallel orientation of the third strand, resulting from Hoogsteen hydrogen bonds between the third strand and the Watson-Crick duplex, is energetically the most favourable while in the T.A*A case the antiparallel orientation of the third strand, resulting from reverse Hoogsteen hydrogen bonds, is energetically the most favourable. These studies when extended to the mixed sequence triplexes, in which the second strand is a mixture of G and A, correspondingly the third strand is a mixture of G and APT, show that though the parallel orientation is still energetically more favourable, the antiparallel orientation becomes energetically comparable with an increasing number of thymines in the third strand. Structurally, for the mixed triplexes containing G and T in the third strand, it is seen that the basepair non-isomorphism between the C.G*G and the T.A*T triplets can be overcome with some changes in the base pair parameters without much distortion of either the backbone or the hydrogen bonds.

Characterization of poly- and monoclonal antibodies to physalis mottle (tymo) virus

Polyclonal antibodies were raised against the Physalis mottle virus (PhMV) and its denatured coat protein (PhMV-P). Analysis of the reactivity of the polyclonal antibodies with tryptic peptides of PhMV-P in dot-blot assays revealed that many of the epitopes were common to intact virus and denatured coat protein. Five monoclonal antibodies to the intact virus were obtained using hybridoma technology. These monoclonal antibodies reacted well with the denatured coat protein. Epitope analysis suggested that probably these monoclonal antibodies recognize overlapping epitopes. This was substantiated by epitope mapping using the CNBr digest of PhMV-P in western blots. All the five monoclonals recognized the N-terminal 15 K fragment. Attempts to further delineate the specific region recognized by the monoclonals by various enzymatic cleavages resulted in the loss of reactivity in all the cases. The results indicate that these monoclonals probably recognize epitopes within the N-terminal 15 K fragment of the coat protein.