Figure 1.
Replication timing profile for chromosome 2L. (A) FACS profile of Drosophila Kc cell synchronization. Asynchronous log-phase Kc cells were treated with the molting hormone Ecdysone to arrest the cells at G2. Following 18-20 h, the cells were washed and resuspended in fresh medium containing HU. The cells accumulated at the G1/S transition within 12 h. Following release from HU, the cells proceeded through S phase. (B) Early and late-replicating sequences were specifically labeled using BrdU. Early or late-replicating sequences were labeled with a 1-h pulse at the beginning or end of S phase, respectively. The BrdU containing DNAs were enriched by immunoprecipitation and differentially labeled with Cy5- or Cy3-conjugated dUTP. (C) Replication timing was determined for each of the 11,243 unique euchromatic sites on chromosome 2L (gray dots) as the ratio of early to late BrdU incorporating sequences. The replication timing profile (black line) was generated by applying a smoothing algorithm to the raw timing data. The numbered cytological intervals of the chromosome are indicated at the bottom.