Replication and functions of macronuclear DNA in synchronously growing populations of Tetrahymena pyriformis

Abstract

It is the objective of this article to discuss aspects of the macronuclear DNA replication and function in heat synchronized populations of Tetrahymena pyriformis. The thymidine analogue 5-bromodeoxyuridine was used as a density marker of synthesized DNA. This density label of DNA, followed by gradient centrifugation, was used to measure DNA replication quantitatively and to gain information about the nature of the DNA synthesis, which takes place at various periods of the cell cycle. Some of the events in the synchronized cell cycle, which are described, may be summarized briefly: After a synchronous cell division the cells initiate a synchronous replication of the macronuclear DNA. The DNA replication begins with replication of the ribosomal genes. Thereafter the bulk nuclear DNA replicates, and parallel with this the genes coding for 5S rRNA and tRNA. The replication of bulk DNA may follow a fixed sequence in each S-period, ensuring full replication of all DNA without double replication of any DNA fraction. However, the replication sequence changes from generation to generation, possibly due to a mixing of the nuclear subnuclei prior to division. The cells appear to have a full complement of genes replicated before the end of the macronuclear S-period. Obligatory DNA synthesis ends and G2-activities are initiated when 70–80% of the DNA has been replicated. the cells are able to divide after the DNA replication has been inhibited in the latter half the preceding S-period. Such cells divide with a fraction of their DNA only partially replicated. This DNA fraction is eliminated from the macronucleus at the time of the division, and it is localized in the cytoplasm during the next cell generation. When the inhibitory treatment is removed at the time of cell division, the extruded DNA continues its replication after the end of the macronuclear S-period. Furthermore, it was found that thymidine starvation, here used as a specific inhibitor of DNA replication, tended to stop replication preferentially in regions of the molecules with relative high dAdT base content and leaving molecules with single stranded regions. Bromodeoxyuridine, which has been a valuable tool in the analysis of the DNA replication in Tetrahymena, is under special circumstances a potent inhibitor of macromolecular synthesis and cell proliferation. Bromodeoxyuridine has to be incorporated into DNA in high amounts before any inhibitory effects are seen. As soon as DNA contains bromodeoxyuridine in sufficient amounts, it will affect transcription, but the various genes respond differently to the substitution. The rDNA, i.e. the genes coding for the two large ribosomal RNA molecules (17S- and 25S RNA) were found to be most sensitive.