Poly(pyrimidine) . poly(purine) tracts have been discovered in the 5'-flanking regions of many eucaryotic genes. They may be involved in the regulation of expression since they can be mapped to the nuclease-sensitive sites of active chromatin. We have found that poly(pyrimidine) . poly(purine) DNAs which contain 5-methylcytosine (e.g. poly[d(Tm5C)] . poly[d(GA)]) will form a triplex at a pH below 8. In contrast, the unmethylated analogue, poly[d(TC)] . poly[d(GA)] only forms a triplex at pHs below 6. Synthetic DNAs containing repeating trinucleotides and poly[d(Um5C)] . poly[d(GA)] behave in a similar manner. Thus the stability of a triplex can be controlled by methylation of cytosine. This suggests a model for the regulation of expression based upon specific triplex formation on the 5'-side of eucaryotic genes.

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.

Both brominated poly[d(GC)] and poly[d(Gm'C)]form stable left-handed Z-DNA structures at physiological ionic strengths. These two antigens were used to prepare monoclonal antibodies frorn immunized mice. The specificity of the antibodies was studied in detail with a solid-phase radioimmune assay as well as by means of competition experiments. Both immunogens produced several relatively nonspecific antibodies but two types of very specific antibody were also distinguished. The first binds poly[d(Gm'C)] but not brominated poly[d(GC)] while the other has the opposite specificity and will only bind the brominated polymer. Z-DNA 8-Bromoguanine S-Methylcytosine Antigenic determinant Monoclonal antibody

No abstract

No abstract

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to BrMRBC. Mitogenic concentrations of Con A prevented the development of both the PFC and TDTH responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and TDTH response against BrMRBC.

Although most duplex DNAs are not immunogenic some synthetic DNAs such as polyl[d(Tm5C)] · poly‐[d(GA)] are weakly immunogenic allowing the production of monoclonal antibodies. The specificity of one of these antibodies, Jel 172, was investigated in detail by a competitive solid‐phase radioimmune assay. Jel 172 bound well to poly[d(TC)]·poly[d(GA)] but not to other duplex DNAs such as poly[d(TTC)] · poly‐[d(GAA)] and poly[d(TCC)]· poly[d(GGA)]. The binding to poly[d(Br5UC)]· poly[d(GA)] was enhanced while that to poly[d(TC)]· poly[d(IA)] was decreased compared to poly[d(TC)]· poly[d(GA)]. Thus, not only is the antibody very specific for a sequence of duplex DNA but it also appears to recognize functional groups in both grooves of the helix.

These studies describe the conditions under which antibody-forming cells and TDTH cells are selectively induced in vitro. TDTH cells are preferentially stimulated when high doses of antigen are included in the culture. Antibody-forming cells, on the other hand, are optimally stimulated with a 100 to 1000-fold less concentration of antigen. The conditions that optimally stimulate TDTH cells also induce a population of suppressor T cells that inhibit the antibody response. However, although their inductive requirements are similar, the suppressor T cells of antibody formation are a distinct subpopulation of cells from the TDTH cells. Whereas the suppressor T cells are LY-1-, 2+, 4-, 6+, and Ia+; the TDTH cells are Ly-1+, 2+/-, 4-, 6+, and Ia-. Furthermore, the DTH cells are sensitive to high doses of irradiation, whereas the suppressor cells are resistant. Based on the Ly phenotype and the kinetics of suppression, the suppressor T cells are not the "feedback suppressors" that have been identified in other systems. The system described in this paper provides a means whereby the cells that regulate humoral and CMI can be studied in vitro.