Cell cycle analysis
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.
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):
The D period is found from the C+D and C period:
D = (C+D) - C
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.
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:
τ = 27 min
ai = 5 min
C = 51 min
D = 25 min
C+D = 76 min