A 95-NM SPACING IN DROSOPHILA POLYTENE CHROMATIN

It has long been debated whether the 30-nm fiber of chromatin is packe d in an orderly array. The fiber may be condensed by supercoiling, pro ducing structures of varying diameter. Alternatively, technical proble ms may have prevented the detection of higher-order structures. We dev eloped a strategy to distinguish between these two possibilities. One potential obstacle to studying the order of packing was the effect of fixatives, dehydrating agents, heat, and embedding polymers on the nat ive structure of chromatin prepared for viewing by electron microscopy . The known tendency of proteins to be degraded by osmium tetroxide an d subsequently to be extracted in the conventional protocols for embed ding might be particularly damaging. To avoid such denaturants and ens ure the retention of proteins in chromatin, the embedding resin HACH w as employed. Drosophila mimica polytene chromosomes were thin sectione d, stained with uranyl acetate, and viewed in the transmission electro n microscope. Images were digitized and subjected to computerized imag e processing. Raw data fries, containing boundary coordinates of all c losed figures in the image, were edited to retain only those regions o f interest (ROIs) that exhibited dimensions similar to those of 30-nm fibers in projection views. Euclidean distances between the centroids of such structures were calculated to obtain linear intercepts between recognizable 30-nm fibers. According to stereology theory, the dimens ions of a lamellar structure can be determined from the volume distrib ution function of such intercepts. Therefore, intercept values were po oled for final data files from five processed images of chromatin. The resulting frequency histogram, showing the number of observations at different intercept values, had a sigmoidal inflection that was diagno stic of a major, new spacing at 95 nm. The 95-nm minimum was sandwiche d between maxima in the 85 to 90 nm interval and throughout the range 105 to 120 nm. The results suggest that established stereological theo ry will be a useful tool for investigating the intractable problem of higher-order chromatin structure.