HYDROXYUREA-INDUCED SYNCHRONY OF DNA-REPLICATION IN THE KINETOPLASTIDA

We have developed a reliable and reproducible method to induce synchro ny of the DNA synthetic cycle in the Kinetoplastida. The method involv es treatment of cultures with 20 mM hydroxyurea (HU) and fetal bovine serum. Both stationary-phase and exponential-phase cultures can be syn chronized. However, in the case of exponential-phase cultures the popu lation doubling time and rate of DNA synthesis of the population influ enced the time of exposure to HU. The treatment of kinetoplastids with 20 mM HU did not adversely affect the cells as judged by oxygen consu mption, RNA, and protein content. We postulate that the requirement fo r high HU levels, which would be toxic to vertebrate cells, may be due to a lower affinity of kinetoplastid ribonucleotide reductase, the ta rget enzyme for HU. Some of the kinetoplastids are pathogens of man an d his food chain. Consequently, the development of a reliable techniqu e for synchronization of the kinetoplastids should not only permit a d etailed analysis of their cellular and molecular biology but provide a means to collect and characterize biochemical and immunochemical subs tances relevant to the infectious process. (C) 1994 Academic Press, In c.