The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A--T and G--C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another.--Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G--C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A--T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A--T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G--C -specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.--The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.

Fluorescence, circular dichroism and sedimentation through cesium chloride gradient techniques were performed to study the physical properties of the binding of the bisbenzimidazole dye Hoechst 33258 (H33258) to natural DNAs and synthetic polynucleotides of defined repeating units. These studies show that Hoechst 33258 exhibits at least two modes of interaction with duplex DNA: (1) a strong base pair specific mode which requires at least 4 consecutive AT base pairs and (2) a weaker mode of binding which is significantly reduced in the presence of high salt (0.4 M NaCl) and exhibits no apparent base specificity. The H33258 binding was found to be sensitive to the substitutions in the minor groove elements of a series of synthetic polynucleotides supporting the model of H33258 binding in the minor groove of the DNA with AT rich sequences. Similar mode of binding was predicted in natural DNAs by methylation of dye-DNA complexes. Footprint analysis of the complex of dye to a pBR322 fragment also supports that a minimum of 4 consecutive AT base pairs are required for H33258 binding to DNA.

Bacteriophage PM2 DNA is a 10‐kb covalently closed circular (ccc) molecule with a reported superhelical density of sigma = ‐0.12. Here we describe the binding of anti‐Z‐DNA antibodies to PM2 form I DNA under high and low salt conditions. The binding to PM2 DNA has been demonstrated by competitive radioimmunoassay (RIA), retardation of the DNA:antibody complexes in agarose gels and visualization by electron microscopy. The antibody binding is dependent on the degree of negative supercoiling. Thus, PM2 form II and form III did not bind the antibody. The low salt RIA results indicated the presence of 200‐400 bp of left‐handed DNA per PM2 molecule. This could reduce the effective superhelical density to sigma = ‐0.04 to ‐0.08, a range comparable with those found for other ccc DNAs in vivo. Electron microscopy revealed that a maximum of 22 antibody molecules bind to PM2. Single‐site restriction with HpaII of the fixed DNA:antibody complex showed a cluster of four to five antibody molecules bound near one end of the linear DNA molecule. The evidence presented indicates that PM2 DNA contains regions of left‐handed conformation under physiological conditions (low salt concentration) as well as at high salt concentrations. In addition, electrophoretic analyses of PM2 topoisomers indicate the presence of left‐handed regions at superhelical densities less than that of isolated PM2 DNA.

Daunomycin and adriamycin were previously found to produce Q-like banding patterns on chromosomes. The interaction of several anthracyclines with both natural and synthetic DNAs and chromosomes has been investigated in more detail. Daunomycin fluorescence is almost completely quenched by natural DNAs with varying base composition from 31 to 72% G-C and by the alternating polymer poly-d(G-C).poly-d(G-C). In contrast, daunomycin fluorescence is quenched by only 50% when the dye interacts with synthetic A-T polymers. Thus, differential quenching of daunomycin fluorescence can account for the production of bright bands at contiguous A-T sequences along the chromosome. Slight differences in fluorescence quenching between the repeating and homopolymeric A-T duplex DNAs were observed which can be attributed to differences in affinity of daunomycin for these DNAs. The aminosugar moiety of daunomycin, daunosamine, increases the binding of daunomycin to DNA and also enhances chromosome banding.--Nogalamycin, which displays no differential quenching with the different DNAs in solution, also fails to produce bands on chromosomes.--These findings suggest that non-random nucleotide sequence arrangements along the chromosome are a basic determinant for dye interaction to produce the observed banding patterns. Specific banding procedures may determine the accessibility of these sites within the chromosomal DNA.