Determining replication timing by time-lapse microscopy
A. Map of the integration sites of tet and lac operators. The replication timing profile of the chromosome region was taken from .
B-E. MATa PGPD1-thymidine kinase (5 copies) PADH1-ENT1 tetR-3CFP GFP-lacI haploid cells with tetO×224 and lacO×256 (as indicated in A: T3765) were arrested by α factor treatment, washed, and released in BrdU-containing medium. After 20 min, samples were collected for time point 0 min, and other time point samples were subsequently collected. B. The budding index and the intensity of each tetOs-1 (red) and lacOs (green) dot of individual cells at different times are shown. Key: dimmer and brighter dots are schematically shown by small and large dots, respectively. The intensity of the dots was investigated in 128-300 cells at each time point. C. BrdU-labelled DNA was amplified by PCR using primers for the ARS (replication origin) and tetOs-1/lacOs integration sites. Throughout the time course, DNA template derived from the same number of cells was used for PCR amplification. If no BrdU was added to the culture there was no increase observed in the PCR products while the budding index increased (data not shown). If cells were treated with HU, there was an increase observed in PCR products for the ARS, but not for the tetOs-1 or lacOs site, while the budding index increased (data not shown). D. Quantification of the intensity of the bands in C. The intensity at time point 0 and the maximum intensity were set to 0 and 100 %, respectively. E. Cellular DNA content was measured by FACS.