Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 itM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Nonimmune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density). Key words: left-handed Z DNA/enhancer/alternating purine-pyrimidine Introduction We have previously developed an electrophoretic assay which detected left-handed Z DNA sites in SV40 DNA (and other circular genomes) using anti-Z DNA IgG binding to negatively supercoiled Form I molecules (Zarling et al., 1984a(Zarling et al., , 1984b. The formation of DNA-IgG complexes depended on external conditions (ionic strength and temperature), nucleotide sequence, the physical state of the DNA (torsional stress) and the anti-DNA immunoglobulin (specificity, valency and concentration). Changes in electrophoretic migration were attributed to the formation of various oligomeric species; the existence of trimers suggested the expression of at least two Z DNA sites per SV40 DNA.In the present study, SV40 Form I DNA-IgG complexes-were preparatively purified by velocity sedimentation using conditions which optimally stabilize natural Z DNA sequences in protein-

We have located an extensive (AC)n-rich but specific sequence downstream of three rainbow trout protamine genes. Although sharing considerable sequence homology, including a perfectly conserved 46 base pair repeat, the sequences exhibit a regular heterogeneity in the length of the (AC)n-rich tracts. Radioimmunoassay experiments, S1 nuclease sensitivity studies, two-dimensional electrophoretic analysis, and immunoelectron microscopy studies have been used to determine if the region could assume a Z DNA conformation. It was found that, in a supercoiled plasmid, the (AC)n-rich region has the ability to attain the Z DNA conformation under physiological conditions.