SLS2016: Cell Cycle Stage Determination of Schizosaccharomyces pombe

This report details an experiment focused on determining the timing of cell cycle stages in Schizosaccharomyces pombe. A standard curve was constructed to correlate DNA content with absorbance, which was then used to estimate DNA concentrations in unknown samples and subsequently infer the durations of the G1, S, and G2 phases. The experiment involved growing S. pombe cells in a synchronous culture, collecting samples at regular intervals, and measuring DNA content using a colorimetric method. Results indicated a relationship between DNA content and cell cycle phase, with estimated timings for G1, S, and G2 phases. The discussion interprets these findings in the context of existing literature, noting the correlation between DNA content, cell number, and cell cycle progression. The maximum DNA concentration was recorded at 380.8 µg•ml-1 and lowest was recorded at 116.8 µg•ml-1. Estimated timings for G1 phase, S phase and G2 phase was at 0 -20 minutes, 20- 40 minutes, and 0 – 20 minutes respectively.

Running head: CELL CYCLE LIFE PROCESS
Determination of the timing of the stages of the cell cycle of the fission yeast
Schizosaccharomyces pombe.
Name of the Student
Name of the University
Author’s Note:

1CELL CYCLE LIFE PROCESS
Abstract
Schizosaccharomyces pombe, a kind of fission yeast, was studied and examined thoroughly in
order learn its cell division, nuclear division and the initiations and controls of DNA
synthesis. in this experiment, a standard curve was constructed with standard DNA content
measuring absorbance at 600 nm and this standard curve was used to determination of DNA
content in unknown samples. Furthermore, G1, S, and G2 phases timing was estimated using
this growth conditions. For the unknown samples maximum DNA concentration was
recorded at 380.8 μg•ml-1 and lowest was recorded at 116.8 μg•ml-1. Estimated timings for
G1 phase, S phase and G2 phase was at 0 -20 minutes, 20- 40 minutes, and 0 – 20 minutes
respectively. Further investigation should be performed with all the DNA, RNA and protein
content as parameters in order to grasp and understand the total coordination.

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Table of Contents
Background:...............................................................................................................................3
Materials and Methods:..............................................................................................................4
Growth medium and Sample preparation:.............................................................................4
Estimation of DNA:...............................................................................................................4
Preparation of standard curve:...............................................................................................5
Experimental procedure:........................................................................................................5
Results:.......................................................................................................................................6
Discussion:.................................................................................................................................8
References:...............................................................................................................................11

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Background:
Schizosaccharomyces pombe, a kind of fission yeast, is a comparatively simple eukaryotic
organism which has been using for to model and study the cell cycles in other eukaryotes
(Asakawa et al. 2014). The experiments were performed to understand the kinetics, control
and timings of the cell cycle event. It has also been found that Schizosaccharomyces pombe
was studied and examined thoroughly in order learn its cell division, nuclear division and the
initiations and controls of DNA synthesis (Kulak et al. 2014). Many different findings have
been reported in this fission yeast cell cycle research area and one of the key findings is that
the nuclear division of this cell is dependent upon the accumulation of critical mass in the
cells. It has been reported that the critical mass increases along with the increment of the
growth of the cells. From the previous it has been established that the cell division of any
organism is dependent on the nuclear division of its cells and a correlation can be drawn in
respect to the mean cell size and growth rate. S phase or beginning of the DNA synthesis is
dependent on the two types of control and the controlling one is the cell size (Hagan, Grallert
and Simanis 2016). Cells have to attain to a certain size before the DNA synthesis can begin
and therefore, it has an extended period of G1 phase or first gap. However, if the daughter
cells already achieved the critical mass, the S phase begins after a small G1 phase instead of
any kind of mass modulated control (Barnum and O’Connell 2014). On the other hand, no
concrete evidence has been reported while establishing the coordination among cell
separation and S phase. Although, some academic research papers suggests that the cells
enters in to the S phase right after the completion of cell separation under certain condition
(Harashima, Dissmeyer and Schnittger 2013).
Therefore, in this experiment, a standard curve will be constructed with standard DNA
content measuring absorbance at 600 nm and this standard curve will be used to

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determination of DNA content in unknown samples. Furthermore, G1, S, and G2 phases
timing will be estimated using this growth conditions.
Materials and Methods:
Growth medium and Sample preparation:
Schizosaccharomyces pombe cells were grown in a synchronous culture. After that a
procedure was performed to select the cell size and cells were collected from the similar
volume of the culture. At the beginning, all the cells were in approximately same size and it
was assumed that they all were in same cell cycle phase.
After that six samples were collected from this synchronous culture in a 2o min interval and
the collected samples were subjected to the following procedures:
i. Homogenisation was performed to lyses the cells Triton containing buffer was for the
treatment.
ii. From the homogenised samples, RNA and protein were removed by incubation with
ribonuclease at 0.1 μg•ml-1 for 1 hour and proteinase K at 0.1 mg/ml.
iii. After that precipitation of nucleic acid have been conducted with the addition of
ethanol in 2:1 proportion.
iv. A buffer at pH 7.5 was used to dissolve the precipitate.
The six samples were labelled as A to F.
Estimation of DNA:
Colorimetric methods were employed for the estimation of the DNA. This estimation was
performed by spectrophotometer. The basis of this method is the detection of colorimetric
change in the solution at the presence of DNA and the colour change occurs due to the
reaction of pentose groups present in the nucleic acid. The links between purines and

5CELL CYCLE LIFE PROCESS
deoxyribose are hydrolysed in the presence of strong acid such as concentrated sulphuric acid
or glacial acetic acid. After that these deoxyribose residues reacts with the diphenylamine
which produces a blue coloration. The absorbance of this blue coloration is maximum at 600
nm and therefore, in this experiment, DNA samples were measured at 600 nm.
Preparation of standard curve:
Standard curve for the DNA samples was constructed by using known samples with DNA
samples ranging from 0 - 400 μg•ml-1. In order to do that, a 400 μg•ml-1 DNA samples were
prepared and dilution were performed using distill water to achieve 5 different concentration
ranging from 0, 100, 200, 300, and 400 μg•ml-1. The dilutions were performed using the
following calculation:
Tube number DNA standard Water
1 0 ml 2.0 ml
2 0.5 ml 1.5 ml
3 1.0 ml 1.0 ml
4 1.5 ml 0.5 ml
5 2.0 ml 0 ml
Absorbance was measured for these 5 standard solutions and a standard curve was calculated
accordingly.
Experimental procedure:
All total 11 samples were measured for absorbance: 5 samples for the creation of standard
curve and 6 unknown samples whose DNA content will be estimated. All the 11 samples
were subjected to the same procedure and the procedure is mentioned below:
i. 4 ml of diphenylamine reagent was added to the each of the 11 samples.
Diphenylamine reagent was prepared by adding 25ml concentrated sulphuric acid to
10 g/l in glacial acetic acid.
ii. After that the samples were mixed gently.

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iii. Samples were placed in the boiling water bath for the duration 10 minutes.
iv. Samples were cooled down after the water bath and absorbance were measured at 600
nm. Sample number 1 was used as blank.
v. A standard curve was constructed from the reading measured for the standard
samples.
Results:
The first step of this experiment was to create a standard curve from the readings collected
against the standard DNA solution. The absorbance readings for the standard DNA solution
are presented in the Table 1.
Table 1: Absorbance readings for the standard DNA solution
Tube No. DNA Conc. (μg•ml-
1) Absorbance600 nm
1 0 0
2 100 0.093
3 200 0.209
4 300 0.289
5 400 0.394
Using the data presented in the Table 1, a standard curve has been constructed and the
standard curve of DNA concentration versus Absorbance at 600 nm is presented in the Figure
1.

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0 50 100 150 200 250 300 350 400 450
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
f(x) = 0.000984 x + 0.000200000000000061
R² = 0.997770037715628
Standard Curve
DNA Concentration (μg•ml-1)
Absorbance600 nm
Figure 1: Standard curve of DNA concentration versus Absorbance at 600 nm
From the standard curve depicted in the Figure 1, the equation Y= 0.001X + 0.0002 has been
generated where Y = absorbance at 600 nm and X = DNA concentration. This equation has
been used to estimate the DNA concentration of six unknown sample. The calculated DNA
concentration of six unknown samples is presented in the Table 2 below.
Table 2: Calculated DNA concentration of six unknown samples
Tube No. Sample No. Absorbance600 nm DNA Conc. (μg•ml-1)
6 A 0.381 380.8
7 B 0.117 116.8
8 C 0.131 130.8
9 D 0.242 241.8
10 E 0.277 276.8
11 F 0.272 271.8
From the data presented in the Table 2, it can be seen that the maximum DNA content present
in the Sample A which is 380.8 μg•ml-1 and lowest DNA content is present in the Sample B

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which is 116.8 μg•ml-1. At the beginning of the experiment, cell number were counted for
each of the unknown samples and these cell numbers can be used to estimate the DNA
content per cell while tallying with the data presented in Table 2. The amount of DNA
present per cell for six unknown samples is presented in the Table 3 below.
Table 3: Amount of DNA present per cell for six unknown samples
Tube No. Sample No. Time
(Mins) Cells/ml DNA Content/Cell (μg) DNA Content/Cell (pg)
6 A 0 8.00E+07 4.76E-06 4.76
7 B 20 150000000 7.79E-07 0.78
8 C 40 150000000 8.72E-07 0.87
9 D 60 150000000 1.61E-06 1.61
10 E 80 150000000 1.85E-06 1.85
11 F 100 300000000 9.06E-07 0.91
* Amount of DNA contents were both presented in Microgram and Picogram in this table.
Data presented in the Table 2 and Table 3 can be used to calculate the timings of G1 phase, S
phase and G2 phase for this organism. The estimated timings of G1 phase, S phase and G2
phase for this organism is presented in the Table 4.
Table 4: estimated timings of G1 phase, S phase and G2 phase for the organism
Schizosaccharomyces pombe.
Phase Name Estimated Timings
G1 0 – 20 minute
S 20 - 40 minute
G2 0 – 20 minute
Discussion:
From the data presented in the above result section it can be said that the there is a strong
correlation between G1 phase, S phase and G2 phase for the organism Schizosaccharomyces

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pombe and the amount of DNA content present in its cells. From the data presented in the
Table 3, it can be seen that amount of DNA content per cell is higher in the earlier stage of
the sample collection and then the amount of DNA content per cell is rapidly decreased for
the next two time duration of sample collection and the amount of DNA content per cell
doubled for the next two sample collection. For the final sample collection, amount of DNA
content per cell decreased significantly again. This suggests that the amount of DNA content
per cell changes with the phase change of the cell cycle. This finding is in line with the
evidence reported in this research area and studies have suggested that DNA content does
change with the phase change during cell growth (Edens et al. 2013). It is also interesting to
find out that the maximum amount of DNA content was noticed during the period where
there is less number of cells present. Amount of DNA content is significantly (~ 4 times) less
while the number cells are at highest. Studies have suggested that the G1 phase is
significantly longer before the start of S phase (Bertoli, Skotheim and De Bruin 2013).
However, in this experiment, it can be seen that it is relatively shorter and duration last only
for 0 to 20 minutes. Although, studies have also reported that the if the daughter cells already
achieved the critical mass, the S phase begins after a small G1 phase instead of any kind of
mass modulated control (Barnum and O’Connell 2014). From the data presented in this
investigation, it can be inferred that this is might be true for this investigation. This deduction
also back up by the findings reported in the Table 2 and Table 3. Table 2 reported that the
maximum DNA content was observed during the G1 phase and from Table 3, it can be seen
that amount of DNA content per cell is also highest during the G1 phase. This higher DNA
content suggests that the cells in G1 phase have already attained the critical mass. The lower
amount of DNA content per cell might be happening due to the DNA replication process in
the S phase.

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Therefore, from the above discussion, it can be concluded that the Schizosaccharomyces
pombe is a good candidate in order learn its cell division, nuclear division and the initiations
and controls of DNA synthesis. It has also been found that there is a strong correlation
between G1 phase, S phase and G2 phase and amount of DNA content present in the cells.
However, studies have also suggested and reported that protein content, RNA content also
plays significant role during the cell growth and in this investigation only amount of DNA
content was studied (Castel and Martienssen 2013). Hence, further investigation should be
performed with all the DNA, RNA and protein content as parameters in order to grasp and
understand the total coordination between G1 phase, S phase and G2 phase and these cell
contents during cell growth.

11CELL CYCLE LIFE PROCESS
References:
Asakawa, H., Yang, H.J., Yamamoto, T.G., Ohtsuki, C., Chikashige, Y., Sakata-Sogawa, K.,
Tokunaga, M., Iwamoto, M., Hiraoka, Y. and Haraguchi, T., 2014. Characterization of
nuclear pore complex components in fission yeast Schizosaccharomyces
pombe. Nucleus, 5(2), pp.149-162.
Barnum, K.J. and O’Connell, M.J., 2014. Cell cycle regulation by checkpoints. In Cell Cycle
Control (pp. 29-40). Humana Press, New York, NY.
Bertoli, C., Skotheim, J.M. and De Bruin, R.A., 2013. Control of cell cycle transcription
during G1 and S phases. Nature reviews Molecular cell biology, 14(8), p.518.
Castel, S.E. and Martienssen, R.A., 2013. RNA interference in the nucleus: roles for small
RNAs in transcription, epigenetics and beyond. Nature Reviews Genetics, 14(2), p.100.
Edens, L.J., White, K.H., Jevtic, P., Li, X. and Levy, D.L., 2013. Nuclear size regulation:
from single cells to development and disease. Trends in cell biology, 23(4), pp.151-159.
Hagan, I.M., Grallert, A. and Simanis, V., 2016. Analysis of the Schizosaccharomyces pombe
cell cycle. Cold Spring Harbor Protocols, 2016(9), pp.pdb-top082800.
Harashima, H., Dissmeyer, N. and Schnittger, A., 2013. Cell cycle control across the
eukaryotic kingdom. Trends in cell biology, 23(7), pp.345-356.
Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N. and Mann, M., 2014. Minimal, encapsulated
proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nature
methods, 11(3), p.319.