Cell cycle analysis: Difference between revisions
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===Determination of initiation age (a<sub>i</sub>) and C+D:=== | |||
[[Image:Theoretical_age_distr.jpg|left|200px]] | |||
From flow cytometry analysis of cells treated with rifampicin and cephalexin (run-out histogram) the proportions of cells that had not initiated replication at the time of drug action (4-origin-cells, streaked) and cells that had initiated (8-origin-cells) can be estimated.The initiation age (a<sub>i</sub>) can be found from the theoretical age distribution described by this formula, | From flow cytometry analysis of cells treated with rifampicin and cephalexin (run-out histogram) the proportions of cells that had not initiated replication at the time of drug action (4-origin-cells, streaked) and cells that had initiated (8-origin-cells) can be estimated.The initiation age (a<sub>i</sub>) can be found from the theoretical age distribution described by this formula, | ||
'''F = 2 - 2 | '''<math>F=2-2^{\frac{(\tau-a_i)}{\tau}}</math>''' | ||
where F is the fraction of cells that had not initiated and τ is the generation time, or from the estimated graph of the theoretical age distribution (streaked portion). | where F is the fraction of cells that had not initiated and τ is the generation time, or from the estimated graph of the theoretical age distribution (streaked portion). | ||
This gives: | |||
'''<math>a_i=\tau-\frac{log(2-F)}{log2}*\tau</math>''' | |||
which is the same as this (log2 is 1): | |||
'''<math>a_i=\tau-log(2-F)*\tau</math>''' | |||
If you have for example a generation time τ=84 minutes and the portion of cells with 4 origins is 66% the formula gives: | |||
'''<math>a_i=84-log(2-0.66)*84=48.5</math>''' | |||
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[[Image:C+D_1.jpg|left|250px]] | [[Image:C+D_1.jpg|left|250px]] | ||
===Determination of the C and D periods:=== | |||
The C period is found from the ''oriC/terC'' ratio obtained by Southern blot or qPCR analysis ([[oriC/ter ratio determination]]) and the generation time (τ): | |||
'''<math>\frac{oriC}{terC}=2^{\frac{C}{\tau}}</math>''' | |||
which gives: | |||
'''<math>C=log_2(\frac{oriC}{terC})*{\tau}</math>''' | |||
The D period is found from the C+D and C period: | |||
'''<math>D = (C+D) - C</math>''' | |||
Example (continues): | |||
C period calculated from the ''oriC/terC'' ratio: 49 min | |||
D period = (C+D) – C | |||
D period = 76 min – 49 min = 27 min | |||
[[Image:C+D_2.jpg|left|250px]] | |||
===The theoretical exponential DNA histogram:=== | |||
A theoretical exponential DNA histogram can be drawn to check whether the obtained values fit with the experimental data. From the C+D period the DNA content of the cells at different time points in the cell cycle can be calculated. | |||
Example: | |||
[[Image:Theoretical_exp_histogram.jpg|left|400px]] | |||
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[[Image:Theoretical_exp_histogram2.jpg|left|200px]] | |||
The individual values of C and D can be varied | |||
to obtain a shape of the theoretical histogram | |||
that gives the best fit to the experimental histogram. | |||
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===Calculation of the average number of replication forks when D=τ:=== | |||
In the example given above, 23% of the cells contain 4 replication forks (4-origin peak in run-out histogram) and 77% contain 12 replication forks (8-origin peak), hence the average number of replication forks in the cell population will be: | |||
(4 x 0.23) + (12 x 0.77) = 10.2 forks | |||
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===Calculation of the average number of replication forks when D≠τ:=== | |||
Example: | |||
4-origin-cells: 23% | |||
8-origin-cells: 77% | |||
τ = 27 min | |||
a<sub>i</sub> = 5 min | |||
C = 51 min | |||
D = 25 min | |||
C+D = 76 min | |||
[[Image:C+D_3.jpg|left|350px]] | |||
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8-origin- | 12 forks → 8-origin peak in run-out histogram = 77% of the cells | ||
6 and 4 forks → 4-origin peak in run-out histogram = 23% of the cells | |||
a<sub> | The fraction of cells containing 6 forks: F = 2 - 2<sup>((τ-a<sub>t</sub>)/τ)</sup> = 2 – 2<sup>((27-2)/27)</sup> = 0.10 | ||
The fraction of cells containing 4 forks: 0.23 – 0.10 = 0.13 | |||
The average number of replication forks: (6 x 0.10) + (4 x 0.13) + (12 x 0.77) = 10.4 forks | |||
[[ | [[Category:Protocol]][[Category:Escherichia coli]] |
Latest revision as of 00:33, 27 August 2013
Cell cycle analysis of Escherichia coli cells
C period = the time for a round of chromosome replication
D period = the time between the end of a round of chromosome replication and cell division
Determination of initiation age (ai) and C+D:
From flow cytometry analysis of cells treated with rifampicin and cephalexin (run-out histogram) the proportions of cells that had not initiated replication at the time of drug action (4-origin-cells, streaked) and cells that had initiated (8-origin-cells) can be estimated.The initiation age (ai) can be found from the theoretical age distribution described by this formula,
[math]\displaystyle{ F=2-2^{\frac{(\tau-a_i)}{\tau}} }[/math]
where F is the fraction of cells that had not initiated and τ is the generation time, or from the estimated graph of the theoretical age distribution (streaked portion).
This gives:
[math]\displaystyle{ a_i=\tau-\frac{log(2-F)}{log2}*\tau }[/math]
which is the same as this (log2 is 1):
[math]\displaystyle{ a_i=\tau-log(2-F)*\tau }[/math]
If you have for example a generation time τ=84 minutes and the portion of cells with 4 origins is 66% the formula gives:
[math]\displaystyle{ a_i=84-log(2-0.66)*84=48.5 }[/math]
The C+D period is estimated from the initiation age (ai), the generation time (τ) and the number of generations spanned per cell cycle.
Example:
4-origin-cells: 23 %
Generation time (τ): 27 min
Initiation age (ai): 5 min
Determination of the C and D periods:
The C period is found from the oriC/terC ratio obtained by Southern blot or qPCR analysis (oriC/ter ratio determination) and the generation time (τ):
[math]\displaystyle{ \frac{oriC}{terC}=2^{\frac{C}{\tau}} }[/math]
which gives:
[math]\displaystyle{ C=log_2(\frac{oriC}{terC})*{\tau} }[/math]
The D period is found from the C+D and C period:
[math]\displaystyle{ D = (C+D) - C }[/math]
Example (continues):
C period calculated from the oriC/terC ratio: 49 min
D period = (C+D) – C
D period = 76 min – 49 min = 27 min
The theoretical exponential DNA histogram:
A theoretical exponential DNA histogram can be drawn to check whether the obtained values fit with the experimental data. From the C+D period the DNA content of the cells at different time points in the cell cycle can be calculated.
Example:
The individual values of C and D can be varied
to obtain a shape of the theoretical histogram
that gives the best fit to the experimental histogram.
Calculation of the average number of replication forks when D=τ:
In the example given above, 23% of the cells contain 4 replication forks (4-origin peak in run-out histogram) and 77% contain 12 replication forks (8-origin peak), hence the average number of replication forks in the cell population will be:
(4 x 0.23) + (12 x 0.77) = 10.2 forks
Calculation of the average number of replication forks when D≠τ:
Example:
4-origin-cells: 23%
8-origin-cells: 77%
τ = 27 min
ai = 5 min
C = 51 min
D = 25 min
C+D = 76 min
12 forks → 8-origin peak in run-out histogram = 77% of the cells
6 and 4 forks → 4-origin peak in run-out histogram = 23% of the cells
The fraction of cells containing 6 forks: F = 2 - 2((τ-at)/τ) = 2 – 2((27-2)/27) = 0.10
The fraction of cells containing 4 forks: 0.23 – 0.10 = 0.13
The average number of replication forks: (6 x 0.10) + (4 x 0.13) + (12 x 0.77) = 10.4 forks