The Journal of Cell Biology, Vol 101, 1422-1433, Copyright © 1985 by The Rockefeller University Press
Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis
DJ Arndt-Jovin, M Robert-Nicoud, P Baurschmidt and TM Jovin
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.
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