Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis

Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution. Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine. These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence. A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images. The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C. thummi; however, preferential binding of one class of antibody over another can be observed in certain regions. These differences can be quantitated by arithmetic division or subtraction of the normalized digital images. If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50%. However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, e.g., A-C. High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D. melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile. Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man. We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences. Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands.