Cell cycle analysis-PDF

Overview

This protocol details the method for determining the initiation age (ai) and C+D period in bacterial cells, as well as the calculation of the C and D periods, the generation of a theoretical DNA histogram, and the calculation of the average number of replication forks.

Determination of initiation age (a_i) 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 (a_i) 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 (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

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

a_i = 5 min

C = 51 min

D = 25 min

C+D = 76 min

1. 12 forks → 8-origin peak in run-out histogram = 77% of the cells
2. 6 and 4 forks → 4-origin peak in run-out histogram = 23% of the cells
3. The fraction of cells containing 6 forks: F = 2 – 2^{((τ-a_t)/τ)} = 2 – 2^((27-2)/27) = 0.10
4. The fraction of cells containing 4 forks: 0.23 – 0.10 = 0.13
5. The average number of replication forks: (6 x 0.10) + (4 x 0.13) + (12 x 0.77) = 10.4 forks