Antibodies to DNA in the left-handed (Z) conformation bind to acid-fixed polytene chromosomes of both Chironomus thummi and Drosophila melanogaster, as shown by direct and indirect immunofluorescence. Comparison of the phase-contrast, immunofluorescence, and DNA staining patterns shows a predominant localization of the antibody to the regions of high contrast and DNA density, the bands. The immunofluorescence is completely abolished by competition with polynucleotides in the Z conformation but not by those in the B form. DNase but not RNase treatment eliminates the antibody staining. Actinomycin D inhibits binding, whereas mithramycin has no effect. The highly reproducible immunofluorescence patterns obtained with the anti-Z-DNA antibodies demonstrate variations in fluorescence intensity between particular bands, which can be quantitated by laser scanning and photon counting techniques. The telomeric regions and DNA strands associated with end-to-end chromosome linkage and ectopic pairing are exceptionally bright. At saturation, average values of 1 IgG molecule per 3,000 base pairs and 1 per 15,000 base pairs are found in the intensely and weakly staining regions, respectively. An alternative statement is that the left-handed Z-DNA conformation is present at a frequency of 0.02-0.1%. The measured differences reflect variations in the local density of Z-DNA sites and not in the affinity for the specific antibody, which appears to be relatively constant throughout the chromosomes (K(d) ≃ 10 nM). These observations taken together with results of biophysical studies on the properties of Z-DNA in solution suggest that regions of DNA in the left-handed conformation could be involved in higher-order structural organization of chromosomes and possibly in modulation of their functional state.
ASJC Scopus subject areas