# Cell cycle analysis

(Difference between revisions)
 Revision as of 03:28, 18 August 2008 (view source)← Previous diff Current revision (02:33, 27 August 2013) (view source) (14 intermediate revisions not shown.) Line 6: Line 6: - '''1.Determination of initiation age (ai) and C+D''':[[Image:Theoretical_age_distr.jpg|left|200px]] + ===Determination of initiation age (ai) 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 (ai) 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 (ai) can be found from the theoretical age distribution described by this formula, - '''F = 2 - 2((τ-ai)/τ)''' + '''$F=2-2^{\frac{(\tau-a_i)}{\tau}}''' + where F is the fraction of cells that had not initiated and τ is the generation time, or from the estimated graph of the theoretical age distribution (streaked portion). where F is the fraction of cells that had not initiated and τ is the generation time, or from the estimated graph of the theoretical age distribution (streaked portion). - + + This gives: + + '''[itex]a_i=\tau-\frac{log(2-F)}{log2}*\tau$''' + which is the same as this (log2 is 1): + '''$a_i=\tau-log(2-F)*\tau$''' + If you have for example a generation time τ=84 minutes and the portion of cells with 4 origins is 66% the formula gives: + '''$a_i=84-log(2-0.66)*84=48.5$''' Line 36: Line 45: [[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): + '''$\frac{oriC}{terC}=2^{\frac{C}{\tau}}$''' + which gives: + '''$C=log_2(\frac{oriC}{terC})*{\tau}$''' + The D period is found from the C+D and C period: + '''$D = (C+D) - C$''' + + 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 - '''2. Determination of the C and D periods:''' + [[Image:C+D_2.jpg|left|250px]] - The C period is found from the ''oriC/terC'' ratio obtained by Southern blot analysis and the generation time (τ): + ===The theoretical exponential DNA histogram:=== - '''''oriC/terC''=2C/τ''' + 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 D period is found from the C+D and C period: + [[Image:Theoretical_exp_histogram.jpg|left|400px]] - '''D = (C+D) - C''' - - 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]] Line 90: Line 105: - '''3. 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. + + + + + [[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. + + + + + + + + + + + + + + + ===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: 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 + + + [[Image:C+D_3.jpg|left|350px]] + + + + + + + + + + + + + + + + + + + + + + + + + + + + 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 + + + [[Category:Protocol]][[Category:Escherichia coli]]

## Current revision

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

## Contents

### 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,

$F=2-2^{\frac{(\tau-a_i)}{\tau}}$

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:

$a_i=\tau-\frac{log(2-F)}{log2}*\tau$

which is the same as this (log2 is 1):

ai = τ − log(2 − F) * τ

If you have for example a generation time τ=84 minutes and the portion of cells with 4 origins is 66% the formula gives:

ai = 84 − log(2 − 0.66) * 84 = 48.5

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):

$\frac{oriC}{terC}=2^{\frac{C}{\tau}}$

which gives:

$C=log_2(\frac{oriC}{terC})*{\tau}$

The D period is found from the C+D and C period:

D = (C + D) − C

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