RNA Metabolism during Regeneration in Stentor coeruleus1

The ciliate protozoan, Stentor coeruleus, has been a favored organism in which to investigate nucleo-cytoplasmic relationships during intracellular regeneration and reorganiza tion. The morphology of stentors and the changes that occur during regeneration and division have been described in detail and were reviewed in Tartar's (1961) treatise. Studies of either regenerative or replicative morphogenesis have largely been concerned with the production and ordering of cortical structures. The available experimental evidence, largely based on unpublished observations of Whiteley (cited in Tartar, 1961) and cyto chemical studies of Weitz (1949) indicate that morphogenesis in stentors involves synthesis of new nucleic acids although Lewis (1962) has reported that no net protein synthesis, but rather interconversion, occurs during regeneration. Whiteley found that stentor regenera tion is inhibited by the purine analog, 8-azaguanine, the pyrimidine analog, 2-thiocytosine, and by ribonuclease (RNase). Weisz (1955) had earlier reported that acriflavin, which inactivates nucleic acid metabolism, also inhibits stentor regeneration. Tartar's (1961, 1963) microsurgical experiments have demonstrated that some function of the macronucleus is essential for regeneration up to the point of formation of the membranellar band primordium, but removal of the macronucleus beyond this point does not hinder the migration and invagination of the cortical structures. Removal of half the macronucleus does not affect the capacity of an individual to regenerate, and even individuals with only a single macronuclear node are capable of regeneration although its onset is retarded by about twenty-four hours. Thus, some quantitative dependence on macronuclear products for regeneration was demonstrated. Evidence from these lines of investigation suggested that synthesis of new RNA in the macronucleus occurs in the early stages of stentor regeneration. Attempts to demonstrate this by conventional autoradiographic methods were frustrated by the relatively large metabolic pool in these organisms. Consequently, RNA metabolism in stentor regeneration was studied by testing the effects on regeneration of substances known to inhibit RNA synthesis, to destroy RNA, or to interfere with its function in protein synthesis. The antimetabolites employed were actinomycin D, which specifically suppresses DNA dependent RNA synthesis according to Reich et al. (1961), 5-fluorouracil (5-FU) a pyrimidine analogue which interferes with principally RNA synthesis and puromycin which Yarmolinsky and De La Haba (1959) found to specifically inhibit protein synthesis RNase was also used as an agent which might be expected to physiologically destroy RNA. These experiments with RNA antagonists were combined with qualitative and semi-quantitative cytochemical investigations of RNA distribution and levels.