332
J . S E E D
acid (RNA plus D N A ) and this can be followed by photometric
measurement of Feulgen stain, to give the relative amount of D N A in
the cell nuclei. Finally, if the cells have been incubated in a radioactive
precursor for a short time before fixation, autoradiographic grain
counts can be made on the cells and correlated with the preceding
physical measurements.
By using successive measurements of this nature in conjunction with
the time-lapse photographic technique it is possible to follow the time
course of the various syntheses during interphase. A randomly growing
culture is photographed at low magnification for approximately one
interphase period at 37°C and is then fixed. From an analysis of the
cine film the various stages in interphase of individual cells in the field
under observation can be found (Walker and Yates, 1952; Seed, 1962).
The subsequent interferometric and photometric measurements on the
individual cell nuclei of known ages then make possible the construction
of curves showing the average increases of nuclear dry mass, D N A +
RNA, and D N A during the interphase cycle o f t h e cell (Seed, 1961,
1962, 1963a).
Although the method is laborious and difficult to execute, it allows
the average synthesis curves to be derived in a relatively direct manner,
and uniformity of growth of the culture and the number of nondividing cells there present can readily be checked from the time-lapse
record. These advantages give the technique considerable superiority
over indirect methods depending on deductions from the value distributions.
In the way outlined above, the cycles of synthesis of D N A and of
nuclear protein were investigated in five types of randomly growing,
rapidly dividing cells in culture (freshly prepared monkey kidney,
human fibroblasts freshly derived from embryo skin, fresh embryomouse fibroblasts, HeLa cells originally derived from a human carcinoma, and L-strain mouse cells, which are capable of producing
tumours in a certain percentage of cases when inoculated into C3H
mice) (Seed, 1961, 1962). For each type of cell, the timing of D N A
synthesis was confirmed by the start of thymidine uptake, as observed
by autoradiography. In all the experiments, nucleoli were excluded
from the interferometric and photometric measurements. Telophase
cells undergoing nuclear reconstruction and early prophase cells were
also not measured, so that changes in nuclear mass associated with
rearrangement of intracellular material in the mitotic process were
excluded from the observations. The results for the normal cells
(monkey kidney, human skin, and mouse fibroblasts) showed that both
the D N A and the majority of the protein contained in the nucleus began
to increase together at the same time after telophase, and thereafter
J . S E E D
acid (RNA plus D N A ) and this can be followed by photometric
measurement of Feulgen stain, to give the relative amount of D N A in
the cell nuclei. Finally, if the cells have been incubated in a radioactive
precursor for a short time before fixation, autoradiographic grain
counts can be made on the cells and correlated with the preceding
physical measurements.
By using successive measurements of this nature in conjunction with
the time-lapse photographic technique it is possible to follow the time
course of the various syntheses during interphase. A randomly growing
culture is photographed at low magnification for approximately one
interphase period at 37°C and is then fixed. From an analysis of the
cine film the various stages in interphase of individual cells in the field
under observation can be found (Walker and Yates, 1952; Seed, 1962).
The subsequent interferometric and photometric measurements on the
individual cell nuclei of known ages then make possible the construction
of curves showing the average increases of nuclear dry mass, D N A +
RNA, and D N A during the interphase cycle o f t h e cell (Seed, 1961,
1962, 1963a).
Although the method is laborious and difficult to execute, it allows
the average synthesis curves to be derived in a relatively direct manner,
and uniformity of growth of the culture and the number of nondividing cells there present can readily be checked from the time-lapse
record. These advantages give the technique considerable superiority
over indirect methods depending on deductions from the value distributions.
In the way outlined above, the cycles of synthesis of D N A and of
nuclear protein were investigated in five types of randomly growing,
rapidly dividing cells in culture (freshly prepared monkey kidney,
human fibroblasts freshly derived from embryo skin, fresh embryomouse fibroblasts, HeLa cells originally derived from a human carcinoma, and L-strain mouse cells, which are capable of producing
tumours in a certain percentage of cases when inoculated into C3H
mice) (Seed, 1961, 1962). For each type of cell, the timing of D N A
synthesis was confirmed by the start of thymidine uptake, as observed
by autoradiography. In all the experiments, nucleoli were excluded
from the interferometric and photometric measurements. Telophase
cells undergoing nuclear reconstruction and early prophase cells were
also not measured, so that changes in nuclear mass associated with
rearrangement of intracellular material in the mitotic process were
excluded from the observations. The results for the normal cells
(monkey kidney, human skin, and mouse fibroblasts) showed that both
the D N A and the majority of the protein contained in the nucleus began
to increase together at the same time after telophase, and thereafter