Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate.
The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS)
Soyer-Gobillard
M O
MO
Département de Biologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique Unité de Recherche Associée 117, Banyuls-sur-mer France.
Géraud
M L
ML
Coulaud
D
D
Barray
M
M
Théveny
B
B
Révet
B
B
Delain
E
E
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex / Q000032:analysis; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA / Q000032:analysis / Q000648:ultrastructure*; D004141:Dinoflagellida / Q000648:ultrastructure*; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009993:Osmium Tetroxide
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007202:Indicators and Reagents; D004247:DNA; D009993:Osmium Tetroxide