Graduate Thesis Or Dissertation
 

Hydroxyurea : physiological effects on Tetrahymena pyriformis and use in cell cycle analysis

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  • Hydroxyurea (10mM) blocks the exponential growth of Tetrahymena pyriformis (GL-I) populations by arresting progress through the cell cycle once the cells enter S-phase. Autoradiographic analysis reveals that HU reduces the uptake of tritiated thymidine (³H-TdR) into macronuclear DNA in S-phase cells to about 1/5 of that in untreated S-phase cells. These slowly synthesizing cells do not divide (even after 10 days) until the HU is removed. The HU-blocked cells are also rescued from S-phase arrest by adding 0. 5mM each of all four DNAprecursor deoxyribonucleosides (XdRs) thus indicating the primary effect of HU is the inhibition of the reduction of ribonucleosides. When the HU-block is removed by either washing the HU from the medium or by rescue with XdRs an excessive delay in recovering the capacity to divide is specifically induced in cells which were in S-phase at time of addition of HU. This delay may be due to the time needed to repair damage to the DNA which was induced specifically during S-phase. Fluoromicroscopic examination of euchrysine-stained cells exposed for a minimum of two hours starting in S-phase show a radical morphological change in the macronucleus. This phenomenon, termed the "halo effect," is characterized by the formation of apparently membrane-associated chromatin aggregates surrounding a constricted chromatin mass. Halo-induction by HU is S-phase specific and upon removal of the HU-block the halo remains until the first recovery division. The halo effect is interpreted as being a morphological indication of the cell's repair response to the damaging effects of HU upon macronuclear DNA. Observations of division of individual cells in microdrops, plus autoradiographic studies using ³H-TdR and standard cell cycle analysis techniques, reveal that HU blocks all cells in the initial 92% of S-phase but does not affect cells in the remaining 8 % of S-phase or in G₂ and division. Thus the fraction of the population of cells that is in G₂ can be approximately determined (within 6 minutes) by the fraction of the population able to divide in the presence of HU. This fraction can be related to the approximate duration of G₂ after mathematically compensating for the age gradient. Thus G₂ analysis of T. pyriformis (GL-I) is accomplished easily, quickly, and inexpensively with the use of HU. The possibilities of utilizing Sphase specific recovery delay and halo induction for simple means of determining the durations of G₁ and S-phase respectively are also discussed. The addition of all four XdRs (0. 5mM) to exponentially growing cells in rich organic complete medium without HU results in the generation time of these cells being decreased by 20%. Cell cycle analysis with phosphorus-33 (³³P) and experiments involving cell phase sensitivity to the presence of excess XdRs show that the excess XdRs are taken up and incorporated or pooled entirely during the initial 50% of S-phase. This acceleration of early S-phase completely accounts for the abbreviated generation time seen under these conditions. When HU is removed after 10 hours of treatment the cells undergo a period of recovery characterized by an increase in the duration of DNA synthesis and in the amount of DNA synthesized before they divide and enter a cell cycle abbreviated at the expense of G₁. These results are related to a model for control of the initiation of DNA replication and cell division based upon the ratio of macronuclear DNA content to cytoplasmic mass.
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