Macronuclear Dna Synthesis in Stentor: Regulation by a Cytoplasmic Initiator* by Noel De Terra Laboratory of Nuclear Medicine and Radiation Biology, Department of Biophysics and Nuclear Medicine, School of Medicine, University of California (los Angeles)

The large ciliate Stentor coeruleus lends itself well to microsurgical procedures. By combining these techniques with autoradiography after exposure of cells to thymidine-H3, it is possible to study mechanisms regulating the occurrence of macronuclear DNA synthesis during the cell growth cycle. Transfer of nuclei between cells which are synthesizing DNA and cells which are not can indicate whether DNA synthesis is determined by the continuous presence or absence of a cytoplasmic factor. Similar experiments involving cell grafting could characterize such a factor as an initiator or an inhibitor. This paper describes these experiments and their results. Previous work on Stentor has suggested that mechanisms regulating the occurrence of several major events in the cell growth cycle are located in the cytoplasm.3-5 The present results will be discussed in terms of these earlier findings and also in relation to current knowledge about regulation of nuclear DNA synthesis. Stentor coeruleus is a large heterotrich ciliate; individual organisms often reach a length of 1 mm when fully extended. Figure 1A shows the main morphological features of S. coeruleus, including the chain macronucleus, the anteriorly placed oral apparatus, and the rows of blue-green pigment granules running longitudinally between the kineties. Throughout interphase, the macronucleus exists as a chain of nodes spiralling almost the entire length of the organism and situated directly beneath the ectoplasm. At 20'C, amitotic division takes approximately eight hours. Tartar has assigned numbers to the stages of amitotic division.25 There are eight stage numbers, each covering a successive one-hour interval so that stage 1 designates the first hour of division while stage 8 refers to the eighth hour, during which cytokinesis occurs. The macronucleus undergoes a series of striking morphological changes during amitotic division. At stage 5, the nuclear nodes begin to coalesce until the nucleus is a compact mass in the center of the cell (stage 6; Fig. 1B); this subsequently elongates into a thin rod (stage 7) which forms nodes again as it is passively pinched in two by the cleavage furrow (stage 8). The cycle of changes in nuclear morphology is associated with the development of new oral structures. It occurs not only during amitotic division but also during oral regeneration, when a new oral apparatus is formed to replace missing or irreparably damaged mouthparts and during physiological reorganization, when new oral structures replace the pre-existing ones for reasons that are not well understood. Spectrophotometric evidence indicates that the macronuclei of ciliates are polyploid.7' 15, 19, 28 Stentors deprived of all but one macronuclear node can regenerate the entire chain and produce viable clones.26 Each node must therefore contain at least one diploid genome and probably contains over a hundred, since ploidy in another large heterotrich, Bursaria truncatella, has been estimated at 2500 X by spectrophotometric measurement of DNA in macroand micronuclei.19