Identification and purification of young macronuclear anlagen from conjugating cells of Tetrahymena thermophila

摘要: Populations of total nuclei collected from growing, starved, and conjugating cells Tetrahymena thermophila have been analyzed by flow microfluorometry. In these analyses, two parameters, size (light scatter) DNA content (fluorescence after staining with ethidium bromide), are evaluated on individual nuclei. With this technique, we identified macro- micronuclei both growing starved shown that contents consistent previously published reports. When in early stages macronuclear anlagen formation (9–10 hr mixing opposite mating types), one major new population is observed which was not seen or cells. These a between very similar to 4C micronuclei. We also class becomes evident precisely as enter stage. later conjugation (16 hr), members disappear corresponding 8C observed. suggest large 4 represent young the process transformation micro-into macronuclei. exploited unique density properties purify Following sedimentation at unit gravity, behave intermediate This fact anterior swell considerably following second postzygotic division approach After contrifugation, less dense than surprising since (2–4C), but larger. Microscopically, relatively unstained methyl green darker using phase contrast either The purity “developmental age” our preparations monitored Routinely, fractions containing 80–90% 2–4C (3–4 × 107) can be obtained 1g centrifugation. As by-products procedures, purified populations obtained. procedures will allow biochemical characterizations made how condensed, transcriptionally inactive, germinal nucleus converted into an extended, active, somatic nucleus.