12.
G E N E T I C
C Y T O L O G Y
421
we work with the log phase in which every cell is undergoing its growth
and reproduction. In such a cell population, the number of cells in
each stage at a given time is roughly proportional to the time required
for completing this particular stage. For example, if mitotic cells
comprise 3-0% of a cell population having a generation time of 12 h,
the time requirement for mitosis would be approximately 12 x 0-03 =
0-36 h, or 21 min (see, however, Chapter 6).
The time requirements for individual stages of mitosis apparently
vary from population to population. Generally, abnormal mitoses,
e.g. those with multipolar spindles, take longer to complete division
than normal figures (Hsu, 1955). Discrepancies in determining mitotic
times are also attributable to the lack of agreement among investigators in delimiting individual phases, especially prophase and telophase (Moorhead and Hsu, 1956).
It was mentioned earlier that microspectrophotometric measurement
suggests that D N A synthesis is performed during interphase. Among
the earlier autoradiographic workers, Howard and Pelc (1953), using
P
32 labeling, confirmed this supposition. They found that cells entering
mitosis during the first few hours after isotope application did not bear
the label. Later, the mitotic cells were labeled. The frequency of labeled
mitoses rapidly increased to reach a peak of nearly 100%. These facts
indicate that D N A synthesis stops a few hours before the onset of
mitosis. This " g a p " period between the completion of D N A synthesis
and mitosis is now known as the G 2 phase. The period of DNA synthesis
is termed the S phase. Another gap period representing the time
between the end of mitosis and the onset of D N A synthesis is termed
the G x phase. Thus the cell cycle can be divided roughly into four
major stages or phases: M (mitosis), G l 3 S, and G 2 , which are cyclical
as are clock-readings.
The application of H
3 - T D R greatly clarifies the timing problem of
DNA synthesis and cell cycle. In essence, it proves, in a more sophisticated manner, the claim made by earlier workers such as Howard and
Pelc. Though parasynchronized populations can also be used if
adjustments are made (Stanners and Till, 1960), the method ideally
applies to logarithmically-growing cell-populations. The reasoning
which supports such an operation is as follows. The cells use thymidine
as a handy precursor of the nucleotide of thymine. If thymidine is
labeled with tritium, the resulting D N A will thus be labeled. The
percentage of labeled cells would theoretically approximate the percentage of cells undergoing D N A synthesis, or those in the S phase. This
is a crude method of estimating the length of the S phase when the
generation time is known. Now if the cell population is labeled with
H
3 - T D R for a short duration, and the cells are harvested for autoradio-
G E N E T I C
C Y T O L O G Y
421
we work with the log phase in which every cell is undergoing its growth
and reproduction. In such a cell population, the number of cells in
each stage at a given time is roughly proportional to the time required
for completing this particular stage. For example, if mitotic cells
comprise 3-0% of a cell population having a generation time of 12 h,
the time requirement for mitosis would be approximately 12 x 0-03 =
0-36 h, or 21 min (see, however, Chapter 6).
The time requirements for individual stages of mitosis apparently
vary from population to population. Generally, abnormal mitoses,
e.g. those with multipolar spindles, take longer to complete division
than normal figures (Hsu, 1955). Discrepancies in determining mitotic
times are also attributable to the lack of agreement among investigators in delimiting individual phases, especially prophase and telophase (Moorhead and Hsu, 1956).
It was mentioned earlier that microspectrophotometric measurement
suggests that D N A synthesis is performed during interphase. Among
the earlier autoradiographic workers, Howard and Pelc (1953), using
P
32 labeling, confirmed this supposition. They found that cells entering
mitosis during the first few hours after isotope application did not bear
the label. Later, the mitotic cells were labeled. The frequency of labeled
mitoses rapidly increased to reach a peak of nearly 100%. These facts
indicate that D N A synthesis stops a few hours before the onset of
mitosis. This " g a p " period between the completion of D N A synthesis
and mitosis is now known as the G 2 phase. The period of DNA synthesis
is termed the S phase. Another gap period representing the time
between the end of mitosis and the onset of D N A synthesis is termed
the G x phase. Thus the cell cycle can be divided roughly into four
major stages or phases: M (mitosis), G l 3 S, and G 2 , which are cyclical
as are clock-readings.
The application of H
3 - T D R greatly clarifies the timing problem of
DNA synthesis and cell cycle. In essence, it proves, in a more sophisticated manner, the claim made by earlier workers such as Howard and
Pelc. Though parasynchronized populations can also be used if
adjustments are made (Stanners and Till, 1960), the method ideally
applies to logarithmically-growing cell-populations. The reasoning
which supports such an operation is as follows. The cells use thymidine
as a handy precursor of the nucleotide of thymine. If thymidine is
labeled with tritium, the resulting D N A will thus be labeled. The
percentage of labeled cells would theoretically approximate the percentage of cells undergoing D N A synthesis, or those in the S phase. This
is a crude method of estimating the length of the S phase when the
generation time is known. Now if the cell population is labeled with
H
3 - T D R for a short duration, and the cells are harvested for autoradio-