Nucleocytoplasmic Ratio Requirements for the Initiation of Dna Replication and Fission in Tetrahymena

Abstract

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S‐phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. 3H‐TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G1. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2–4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post‐fission‐daughter cells and pre‐fission cells is identical (standardized to N/C = 1) but late G1 cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4× and the C continues to increase until just prior to recovery division when it also reaches a value 4× that of the original daughter cells. Thus, the N/C = 1 is re‐established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G1. The elimination of G1 (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.

Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants.

“…Since the macronucleus is engaged exclusively in current service to the cytoplasm, it is not surprising that its replication should somehow be controlled by a size-related feedback mechanism. Thcre are several types of experimental evidence that such a feedback mechanism does exist in ciliates (29,47,66).…”

“…7 and 8 underestimate the total increase in macronuclear DNA content in dividing wild-type cells, as the DNA that enters the macronuclear extrusion bodies formed during macronuclear division is not represented. However, extrusion bodies make up a small portion of the total macronuelear DNA in exponentially dividing amicronucleate tetrahymenas (8,66), and observations by Doerder and DeBault, and by Nanney (see reference 12, p. 486), indicate that DNA extrusion occurs infrequently during macronuclear division in exponential phase syngen 1 cells. number increased very little during the first 2 h after the temperature shift, while macronuclear DNA content increased rapidly from the start.…”

MYO1, a Novel, Unconventional Myosin Gene Affects Endocytosis and Macronuclear Elongation inTetrahymena thermophila

“…Tetrahymena is known to regulate DNA content by altering both the timing and the extent of DNA synthesis. If DNA content is too low, cells go through additional DNA synthesis prior to G2 (Worthington et al 1976), and if DNA content is too high, an S period will be followed by two consecutive cell divisions (Doerder and DeBault 1978). Release of chromatin extrusion bodies may also regulate DNA content (Cleffmann 1980).…”

Analysis of a mutant expressing temperature‐sensitive changes of cell size in Tetrahymena

“…These models hold that the cell not only has to attain a certain critical cell size as a prerequisite for cell division but also that arrival at this threshold is sufficient to initiate division (Johnston et al, 1979; Killander and Zetterberg, 1965a,b;Nurse, 1975;Fantes and Nurse, 1977;Prescott, 1960). Since there is a close correlation between ploidy and cell size the parameter effective in the control of cell-cycle events may well be related to the nuclear cytoplasmic ratio (Berger, 1982;Rasmussen and Berger, 1982;Worthington et al, 1976).These models are challenged by the fact that in Tetrahymenu the relationship between cell size and division is uncoupled in the heat-shock synchronisation treatment (Zeuthen and Rasmussen, 1972). Furthermore, recently it was shown that cell division can be induced in the absence of cell growth in mouse 3T3 cells (Zetterberg and Engstrom, 1984), indicating that neither size per se nor increase in mass is an indispensable feature for the initiation of cell division.…”

“…These models hold that the cell not only has to attain a certain critical cell size as a prerequisite for cell division but also that arrival at this threshold is sufficient to initiate division (Johnston et al, 1979;Killander and Zetterberg, 1965a,b;Nurse, 1975;Fantes and Nurse, 1977;Prescott, 1960). Since there is a close correlation between ploidy and cell size the parameter effective in the control of cell-cycle events may well be related to the nuclear cytoplasmic ratio (Berger, 1982;Rasmussen and Berger, 1982;Worthington et al, 1976).…”