# Cell cycle analysis

(Difference between revisions)
 Revision as of 04:36, 18 August 2008 (view source)← Previous diff Revision as of 04:43, 18 August 2008 (view source)Next diff → Line 95: Line 95: Example: Example: + + [[Image:Theoretical_exp_histogram.jpg|left|400px]] + Line 124: Line 127: [[Image:Theoretical_exp_histogram2.jpg|left|200px]] [[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. + + + + + + + + + + + + + + + '''4. 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 + + + + '''5. 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

## Revision as of 04:43, 18 August 2008

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

1.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((τ-ai)/τ)

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).

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

2. Determination of the C and D periods:

The C period is found from the oriC/terC ratio obtained by Southern blot analysis and the generation time (τ):

oriC/terC=2C/τ

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

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.

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.

4. 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

5. 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