Cell cycle studies in Paramecium : effects of abrupt changes of nutritional state on cell cycle regulation
Creators: name: Ching, Ada Sik-Lun
Publisher: name: University of British Columbia
Year: 2010

description: The controls over initiation of DNA synthesis, initiation of cell division, regulation of macronuclear DNA content, and the relationship between cell mass and growth rate were examined in cells growing under nutrient constraint, or in cells experiencing a change in growth conditions through nutritional enrichment (shift-up) or nutritional shift-down. Reduction in both cell mass and DNA content was achieved by growing Paramecium cells under nutritional limitation in the chemostat. Under the extreme condition in the chemostat, the normally balanced relationship between DNA content and cell mass (Berger, 1984 Kimball, 1967) is uncoupled. The DNA content in these cells is maintained at about 50 units, but cell mass can be as little as 24% of normal. The generation time in these slow growing cells was increased 4 to 5 times that of rapidly growing cells; the growth rate was also reduced by about the same proportion. Nutritional shift-up was done by transferring the chemostat cells to medium of excess food. Similarly, nutritional shift-down was performed by transferring cells either to the chemostat or to exhausted medium. The timing of DNA synthesis initiation is largely determined in the preceding cell cycle. Although growth rate (protein synthesis rate) responds quickly to the new conditions, the timing of DNA synthesis initiation is not readjusted immediately and reflects that of the parental cell cycle. The rate at which cells enter S phase however, is affected by a reduction in growth rate. The criteria for DNA synthesis initiation are not determined by cell mass per se. First, cell mass increases to about 180% of the initial G1 value at the time of DNA synthesis initiation following a nutritional shift-up. This value is much greater than that of well-fed controls (118%). However, the increase in cell mass up to the mean time of DNA synthesis initiation and cell division are not significantly different than that observed in well-fed cells. This suggests a mass-related control over initiation of DNA synthesis. Second, cells initiate DNA synthesis even when there is a net decrease in cell mass following nutritional shift-down. Thus, an increase in cell mass per se is not necessary for DNA synthesis initiation. Unlike initiation of DNA synthesis, the regulatory mechanisms determining the macronuclear DNA synthesized reflects solely the current nutrient conditions. Cells in chemostat culture normally maintain about half the normal amount of DNA (about 50 units). Following nutritional shift-up cells synthesize 100 units of DNA instead. Similarly cells synthesize only 50 units of DNA following nutritional shift-down. The amount of DNA synthesized, therefore, is related to the growth rate, and as discussed later, is also related to the commitment point to cell division. This study also reveals that the point of initiation of cell division is not time-dependent. It does not occur at a fixed duration following the previous fission or the initiation of DNA synthesis. The point of commitment to division occurs at about 95 minutes before fission regardless of growth rate. Analysis of the effects of macronuclear DNA synthesis inhibition in cc1 cells after the transition point for division indicate that cells synthesize 50 units of DNA before the point of commitment to division. This suggests that cells are committed to divide after synthesizing about 50 units of DNA. Following this point, rapidly growing cells will produce 50 units of DNA before fission; whereas slow growers will accumulate an amount proportional to their growth rate. There are reasons to believe that the threshold value of DNA for commitment to cell division may be 41 units instead of 50.