A psoralen crosslinking assay was utilized to detect localized, unrestrained DNA supercoiling (torsional tension) in vivo in Drosophila chromosomal regions subject to differential transcriptional activity. By comparing rates of crosslinking in intact cells with those in cells where potential tension in chromosomal domains was relaxed by DNA strand nicking, the contribution to psoralen accessibility caused by altered DNA‐protein interactions (e.g. nucleosomal perturbations) was distinguished from that due to the presence of unrestrained supercoiling in a region of interest. The heat shock protein 70 (hsp70) genes were wound with a significant level of superhelical tension that remained virtually unaltered whether or not the genes were transcriptionally activated by thermal elevation. Constitutively expressed 18S ribosomal RNA genes also exhibited unrestrained superhelical tension at a level comparable with that across hsp70. In contrast, flanking regions downstream of each of the divergent hsp70 genes at locus 87A7 exhibited substantially less tension. Thus the results point to the existence of stable, torsionally stressed topological domains within eukaryotic chromosomal DNA, suggesting that the relaxing action of topoisomerases is not ubiquitous throughout the nucleus but, in fact, is likely to be tightly regulated.

Background: Chronic arsenic exposure is a worldwide health problem. How arsenic exposure promotes a variety of diseases is poorly understood, and specific relationships between experimental and human exposures are not established. We propose phenotypic anchoring as a means to unify experimental observations and disease outcomes.Objectives: We examined the use of phenotypic anchors to translate experimental data to human pathology and investigated research needs for which phenotypic anchors need to be developed.Methods: During a workshop, we discussed experimental systems investigating arsenic dose/exposure and phenotypic expression relationships and human disease responses to chronic arsenic exposure and identified knowledge gaps. In a literature review, we identified areas where data exist to support phenotypic anchoring of experimental results to pathologies from specific human exposures.Discussion: Disease outcome is likely dependent on cell-type–specific responses and interaction with individual genetics, other toxicants, and infectious agents. Potential phenotypic anchors include target tissue dosimetry, gene expression and epigenetic profiles, and tissue biomarkers.Conclusions: Translation to human populations requires more extensive profiling of human samples along with high-quality dosimetry. Anchoring results by gene expression and epigenetic profiling has great promise for data unification. Genetic predisposition of individuals affects disease outcome. Interactions with infectious agents, particularly viruses, may explain some species-specific differences between human pathologies and experimental animal pathologies. Invertebrate systems amenable to genetic manipulation offer potential for elaborating impacts of specific biochemical pathways. Anchoring experimental results to specific human exposures will accelerate understanding 
of mechanisms of arsenic-induced human disease.

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Thiols react at room temperature in dilute solution with 8-azidoadenosine and its nucleotides to give the corresponding 8-aminoadenosine derivatives. The reaction which takes place in the dark is base-catalysed and is particularly rapid when dithiols, e.g. dithiothreitol are used.

Methidiumpropyl-EDTA-iron(II) [MPE-Fe(II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE-Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE-Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE-Fe(I) and micrococcal nuclease cleavage. MPE-Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some doublestranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE-Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE-Fe(H) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE-Fe(ll) will be very useful in the analysis of chromatin structure.With our current appreciation of the nucleosome as the fundamental unit of chromatin condensation (1-3), it has been pertinent to ask whether or not there is a functional requirement for a particular nucleosomal array. This aspect of chromatin structure has been most often expressed in the concept of specific nucleosome positioning (or "phasing") at a few or many loci of the eukaryotic genome, perhaps in a cell-, tissue-, or development-specific manner. Possible advantages of such positioning have been envisaged by many investigators, although no positive evidence for its actual functional importance in vivo has yet been presented. Numerous studies arguing for a specific or, conversely, for a random distribution of nucleosomes have been reported, and these have been reviewed (4-7). Many of these experiments have utilized micrococcal nuclease for generation of nucleosomal arrays. The DNA is purified subsequent to the nuclear digestion and the cleavage sites are mapped by reference to well-characterized restriction sites. Unfortunately, micrococcal nuclease has a marked sequence preference and introduces cleavages into purified DNA at quite specific and reproducible positions (8,9). In some cases these occur at exactly the same sites in chromatin, leading to uncertainty concerning which is chromatin specific and which is purely sequence specific....

We investigated in detail the structural changes that occur in nuclear chromatin upon activation of the four small heat shock protein genes in D. melanogaster. Both -the chemical cleavage reagent methidiumpropyl-EDTA-iron(II) [MPE Fe(II)] and the nuclease DNase I revealed a complex pattern of four or five hypersensitive sites upstream of each gene before activation. In addition, MPE-Fe(II) detected a short positioned array of nucleosomes located on each coding region. Upon heat shock activation a number of changes in the patterns occurred. For each gene, at least one of the upstream hypersensitive regions was eliminated or substantially shifted in position. Regions were established which became highly refractile to digestion by either MPE -Fe(II) or DNase I and, as such, appeared as small "footprints" in the pattern. The location of these refractile regions relative to the cap site varied for each gene examined. The coding regions themselves became highly accessible to DNase I. The nucleosomal arrays detected by MPE-Fe(II) were characterized by a considerable loss of detail and significantly enhanced accessibility, the extent of which probably reflected the relative transcription rate of each gene. Careful mapping of the location and extent of each upstream footprint and comparison with the DNA sequence revealed the presence at each location of two (or more) contiguous or overlapping segments that bear high homology to the heat shock consensus sequence C-T-N-G-A-A-N-N-T-T-C-N-A-G. A specific protein factor (or factors) is most likely bound at or near these sequences in heat-shocked Drosophila cells.

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