In Acanthamoeba, two different cell types are known. Trophozoites are generated in the mitotic division cycle, whereas cells committed at late G2 phase of the cell cycle develop into cysts in response to starvation. In this paper we study the role of timing of DNA replication in regulating development. The investigation was performed with cultures growing in a non-defined medium (ND cells) that show a high encystation competence and with cultures that have been growing in a chemically defined medium (D cells) for several years and show a low encystation competence. Bivariate DNA/BrdUrd distributions show that ND cells progress through a cycle in which the short replication phase occurs immediately and exclusively after prior completion of mitosis. These cells arrest at late G2 phase of the cell cycle during the stationary stage. In D cells, DNA replication and mitosis seem to be uncoupled, since replication takes place before as well as after mitosis. These cells arrest within their replication phase during the stationary stage. These findings indicate that D cells do not progress into late G2 phase of the cell cycle and hence do not have the competence for commitment. The alternate timing of DNA replication and the low encystation competence of D cells can be reversed by cultivation of these cells in ND medium. Synchronization experiments reveal that late G2 phase ND cells exhibit a low capacity for BrdUrd incorporation and growth after transfer into D medium, whereas ND cells of earlier phases of the cell cycle show premitotic incorporation of BrdUrd into nuclear DNA and growth. These findings suggest on the one hand that premitotic DNA synthesis is a prerequisite for growth of cells in D medium, and that there is a dependence of the induction of premitotic DNA synthesis on the cell cycle, and on the other hand that a reciprocal relationship exists between the capacity of premitotic DNA synthesis and commitment to differentiation.

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