Abstract. The usual conformation of DNA is a righthanded 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. Mclntosh, 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 aldehydeprefixed, 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-rim gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their archshaped'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 fibriUo-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. We also demonstrate the preservation of antibody activity on DNA molecules preincubated in OsO4 solutions. The role of the Z-DNA conformation as a possible site for unwinding and DNA processing in chromosomes that lack nucleosomes and that are permanently condensed is discussed. DNOFLAGELLATE protists are primitive eukaryotes, as demonstrated by ultrastructural, biochemical, and molecular biological studies (25,27...

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The Lsp-2 gene encodes a major larval serum protein (hexamerin) of Drosophila melanogaster: Transcription of Lsp-2 is controlled by 20-hydroxyecdysone. Here we report the analysis of the structure of the Lsp-2 gene including the adjacent 5' and 3' sequences. In contrast to all other known hexamerin genes, Lsp-2 does not contain an intron. The Lsp-2 mRNA measures 2312 bases, as deduced from experimental determination of the transcription-start and stop sites and conceptual translation results in a 718 amino acid hexamerin subunit, including a 21-amino-acid signal peptide. While the calculated molecular mass of the native 697-amino-acid subunit is 83.5 kDa, mass spectrometry gave a value of 74.5 kDa. We detected in the Lsp-2 gene a 2052-bp antisense ORF that probably does not code for any protein. An unusual accumulation of rarely used codon triplets was found at the 5' and 3' ends of the Lsp-2 OW.The calculated secondary structure matches well with that of arthropod hemocyanins. Electron micrographs show for LSP-2 hexamers a cubic shape, which can not be easily reconciled with its hexameric structure. Phylogenetic analysis revealed that LSP-2 diverged from the LSP-1 -like hexamerins after separation of the Diptera from other insect orders.