Cytoplasmic Dna Synthesis

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detcctable structures. Staining experiments with basic dyes, including the fluorochrome acridine orangc, revealed the presence of large numbers of 0.3 to 0.5 # particles in the cytoplasm of all cells studied. The cffcct of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with Ha-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles. Extranuclear DNA as a normal cell component has been reported in a variety of biological systems. In several instances the DNA is associated with presumably autonomous or semiautonomous cytoplasmic organelles. The DNA's of the kinetoplast of trypanosomes (1), of proplastids (2), and of chloroplasts (3, 4) fall into this category. In other cases, however, normal cytoplasmic location of DNA has not been related to a recognizable structure. These include the DNA in the cytoplasm of amphibian eggs (5, 6) as well as the presumptive DNA's reported in the cytoplasm of lily anthers (7), Tetrahymena (8), fern eggs (9), and Amoeba proteus (10, 11). This second group of cytoplasmic DNA's raises the question whether or not DNA or a DNA-like polymer can in fact be synthesized and maintained in a cell in the absence of structural organization beyond the molecule itself. The possibility of such synthesis is made plausible by the in vitro synthesis of deoxyribonucleotide polymers, with or without priming molecules, by means of the polymerase reaction (12, 13), as well as by the demonstration of polymerase activity in the cytoplasm of mammalian ceils (14) and its speculatively inferred presence from less direct experiments in the cytoplasm of Amoeba proteus (15). The present report deals with studies undertaken to learn whether or not cytoplasmic incorporation of tritiated thymidine into a deoxyribonuclease-sensitive molecule in Amoeba proteus is associated with a DNA-containing cell component with microscopically detectable structure. The application of cytochemical techniques, coupled with autoradiography and centrifugal stratification of living cells, has led to evidence which indicates that most if not all cytoplasmic thymidine incorporation into this organism occurs in or on discrete particles which can be resolved with light microscope optics but are below 1 525 on July 3, 2017 jcb.rress.org D ow nladed fom m i c r o n in d i a m e t e r w h e n visual ized wi th s t a n d a r d basic dyes or w i t h the f l uo roch rome ac r id ine