The Inifiation, Maintenance and Termination DNA Synthesis: A Study of Nuclear DNA Replication Using Amoeba Proteus As a Cell Model

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Abstract
By means of the nuclear transfer technique for amoeba, combinations of nuclei and cytoplasma from all parts of the cell cycle were available for examining the individual roles of the nucleus and cytoplasm in nuclear DNA replication. Neither S-phase nor division sphere cytoplasm proved capable of initiating a new round of nuclear DNA synthesis in the G2 nucleus. There was some indication that G2 nuclei which were transferred into early prophase cells, i.e. before the formation of a regular division sphere, did incorporate more [3H]thymidine than control G2 nuclei. Positive proof of the induction of DNA synthesis in ‘immature’ nuclei was observed in only two cases. When young G2 nuclei were transplanted into late G2 amoebae, the addition of the donor nucleus generally resulted in the older nucleus being held in a late G2 phase until the younger nucleus passed through its G2. Division of 90% of heterophasic homokaryons was synchronous, with a subsequent synchrony of DNA synthesis. A study of variance in [3H]thymidine incorporation by S nuclei sharing the same cytoplasm - using binucleate, trinucleate and multinucleate homokaryons - showed that nuclei through the peak-S period synthesized DNA at approximately similar rates. The large differences in [3H]thymidine incorporation by nuclei of amoebae of equal age appear due to differences in endogenous precursor pools. These would vary both with differences in food intake and with the draining of remote precursor pools for simultaneous cellular activities, particularly RNA synthesis. When sharing the same cytoplasm nuclei in peak S incorporated similar amounts of [3H]thymidine. Though cytoplasm did not influence the progress of DNA replication by a nucleus, it did influence the use of exogenous [3H]thymidine by the cell, and in so doing caused much of the variation observed in the labelling of nuclei during S. Nuclei sharing the same cytoplasm, and so subject to the same precursor pool changes, incorporated similar amounts of exogenous thymidine. Once DNA synthesis had been initiated it continued to completion regardless of the cytoplasm which surrounded it. Thus neither the maintenance nor termination of DNA synthesis required a special cytoplasmic state.

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