# Cell cycle analysis

### From OpenWetWare

Line 156: | Line 156: | ||

(4 x 0.23) + (12 x 0.77) = 10.2 forks | (4 x 0.23) + (12 x 0.77) = 10.2 forks | ||

+ | |||

+ | |||

Line 179: | Line 181: | ||

[[Image:C+D_3.jpg|left|350px]] | [[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<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 |

## Revision as of 03:51, 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 (a**:

_{i}) and C+DFrom 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_{i}) 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 (a_{i}), 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 (a_{i}): 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*=2^{C/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

a_{i} = 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