DNA in eukaryotic chromosomes is organized into topological domains of supercoiled DNA. In prokaryotes much of this supercoiling is equilibrated as unrestrained torsional tension in the DNA helix. In bacteria this torsional strain has been shown to be important in mechanisms controlling gene expression. Although eukaryotic DNA is supercoiled, most supercoils are restrained in nucleosomes and, on average, torsional tension is not equilibrated with the DNA helix. Although there is no direct evidence, current models for gene regulation in eukaryotes invoke a torsionally strained helix which could promote breathing of the DNA or formation of left-handed Z-DNA. The present in vivo assay for unrestrained torsional tension involves measurement of rates of photobinding of 4,5',8-trimethylpsoralen to DNA. This procedure provides averaging measurements which would not detect torsional tension if present in a small fraction of the chromosome, such as in active genes. This assay will be modified to quantitate rates of psoralen cross-linking to selected restriction fragments of DNA as an indication of torsional strain. An Exonuclease III-photoreversal assay will be developed to quantitate psoralen binding at the level of base pair resolution. These assays will then be applied in vivo to determine if selected regions of SV40 minichromosomes or Drosophila heat shock genes are wound with unrestrained torsional tension. In addition, we will apply recently developed in vivo assays for cruciform and Z-DNA formation as in vivo probes for unrestrained tension. Potential cruciform and Z-DNA forming sequences that form alternate conformations at precise levels of supercoiling will be synthesized. Insertion of these sequences into SV40 DNA and near Drosophila heat shock genes via P-element transformation will provide torsionally tuned probes for various levels of unrestrained DNA supercoiling in vivo.