12. G E N E T I C C Y T O L O G Y
429
50% or thereabouts, really does not confer much usefulness to the
worker for whatever he is investigating. As a matter of fact, mitotic
synchrony produced by such physical techniques is usually very low.
One method, successfully employed with HeLa cells grown in monolayer cultures, is to dislodge mitotic cells differentially and use them for
fresh subcultures (Terasima and Tolmach, 1961). Methods of this type
seem to be most desirable since physiological conditions are not
seriously distorted.
Chemical methods of synchronizing divisions in mammalian cells
appear to offer more hope than the broad physical changes, because
agents which interfere with one specific enzymatic reaction can be
chosen while other enzymes are not affected. It has been a current
practice to use agents that cause thymine deficiency for synchronizing
mitosis. Rueckert and Mueller (1960) used two such compounds, the
folic acid analogue amethopterin (4-amino-JV
10
-methyl folic acid),
and an inhibitor of thymidylate synthetase, F U D R , to test the reaction
of HeLa cells in respect to cell division and kinetics of macromolecular
synthesis. When thymine metabolism is inhibited, D N A synthesis stops.
After an appropriate period of such an unbalanced growth, thymidine
is introduced to the medium to reverse the inhibition. There is no
necessity to remove the inhibitor in this procedure. The cells immediately utilize the supplied thymidine and resume D N A synthesis.
Presumably the mitosis of the cells is synchronized. An illustrative
example from these authors is presented in Fig. 8, which demonstrates
that no increase of cell number was observed for 16 h in the presence
of amethopterin; but 6-8 h after the thymidine reversal the cell number
practically doubled.
It appears that the duration of thymine starvation is important. If
it is not long enough, synchronization will be ineffective; if too long,
irreversible damage to the cell populations results in a rapid death of
the cells. Biological response to such treatment was elucidated by
Mueller, Kajiwara and Stubblefield (1961) and Stubblefield (1961),
again using HeLa cells. Presumably, when the inhibitors are applied,
DNA synthesis ceases immediately. Cells in the G x phase remain in the
G x phase, probably accumulating just prior to the onset ofthe S phase.
The G 2 cells and the M cells, with no need of thymidine at the moment,
move regularly until they, too, reach the threshold ofthe S phase. Cells
in the S phase, however, behave differently. When the agent is applied,
some cells are near the end of the S phase; some are at the middle of
that period; and some just begin to synthesize DNA. Since their
thymine metabolism is stopped, they remain at their respective individual substages of the S phase. When the reversal agent is added, all
the cells previously stopped at the end stage of the G a phase simultan-
429
50% or thereabouts, really does not confer much usefulness to the
worker for whatever he is investigating. As a matter of fact, mitotic
synchrony produced by such physical techniques is usually very low.
One method, successfully employed with HeLa cells grown in monolayer cultures, is to dislodge mitotic cells differentially and use them for
fresh subcultures (Terasima and Tolmach, 1961). Methods of this type
seem to be most desirable since physiological conditions are not
seriously distorted.
Chemical methods of synchronizing divisions in mammalian cells
appear to offer more hope than the broad physical changes, because
agents which interfere with one specific enzymatic reaction can be
chosen while other enzymes are not affected. It has been a current
practice to use agents that cause thymine deficiency for synchronizing
mitosis. Rueckert and Mueller (1960) used two such compounds, the
folic acid analogue amethopterin (4-amino-JV
10
-methyl folic acid),
and an inhibitor of thymidylate synthetase, F U D R , to test the reaction
of HeLa cells in respect to cell division and kinetics of macromolecular
synthesis. When thymine metabolism is inhibited, D N A synthesis stops.
After an appropriate period of such an unbalanced growth, thymidine
is introduced to the medium to reverse the inhibition. There is no
necessity to remove the inhibitor in this procedure. The cells immediately utilize the supplied thymidine and resume D N A synthesis.
Presumably the mitosis of the cells is synchronized. An illustrative
example from these authors is presented in Fig. 8, which demonstrates
that no increase of cell number was observed for 16 h in the presence
of amethopterin; but 6-8 h after the thymidine reversal the cell number
practically doubled.
It appears that the duration of thymine starvation is important. If
it is not long enough, synchronization will be ineffective; if too long,
irreversible damage to the cell populations results in a rapid death of
the cells. Biological response to such treatment was elucidated by
Mueller, Kajiwara and Stubblefield (1961) and Stubblefield (1961),
again using HeLa cells. Presumably, when the inhibitors are applied,
DNA synthesis ceases immediately. Cells in the G x phase remain in the
G x phase, probably accumulating just prior to the onset ofthe S phase.
The G 2 cells and the M cells, with no need of thymidine at the moment,
move regularly until they, too, reach the threshold ofthe S phase. Cells
in the S phase, however, behave differently. When the agent is applied,
some cells are near the end of the S phase; some are at the middle of
that period; and some just begin to synthesize DNA. Since their
thymine metabolism is stopped, they remain at their respective individual substages of the S phase. When the reversal agent is added, all
the cells previously stopped at the end stage of the G a phase simultan-