As expected, most of the DNA replication genes were induced in late GS phase.Interestingly, many of the DNA repair, checkpoint, and mitotic genes were also induced in late G and S phase, suggesting that EF could link expression of DNA replication and repair genes and play a role in the activation of genes required not only during S phase but at subsequent stages of the cell cycle. We observed striking enrichment of EF on each of the genes we examined in quiescent WI cells.Interestingly, several DNA repair genes were highly enriched with EF antibodies; the enrichment was comparable to, or exceeded, that observed with the previously characterized cell cycle regulators, p and cdcA. As for other mammalian cell cycle synchronization techniques, it is not feasible to obtain a homogeneous population of quiescent WI cells, and the presence of residual cycling cells results in the detection of EF at this stage.The observed connection between EF and a significant number of DNA repair genes as well as genes involved in the DNA <a href="https://www.ncbi.nlm.nih.gov/pubmed/17157884">buy
Varenicline Tartrate</a> damage and spindle checkpoints was unexpected.In some cases, genes clustered into two or more functional categories.We tested this idea by synchronizing wildtype and p; p mouse embryonic fibroblasts by serum starvation and restimulation and then harvesting cells at various points after restimulation.We isolated RNA and assessed the level of each transcript by reverse transcription coupled with the polymerase chain reaction. Novel EF targets identified in this study are derepressed in mouse embryonic fibroblasts lacking p and p.Cells were harvested at various times after restimulation, as indicated, and RNA was isolated.RTPCR analysis was performed using primers corresponding to selected DNA repair, checkpoint control, and mitotic genes as shown.Cyclin A and actin serve as positive and negative controls for cell cycle synchronization and RNA normalization, respectively.This result, when combined with our FACS analysis, also confirms that these cells were synchronized effectively. Importantly, transcription of a control gene, actin, was not affected by loss of p and p.We examined genes involved in DNA repair, mitotic and DNA damage checkpoints, and mitosis.Remarkably, we found that loss of p and p led to dramatic derepression in GG of each of the EF targets that we identified, although the degree of derepression varied from one gene to another.The DNA repair gene RADL, mitotic checkpoint genes MADL and TTK, and the HEC and NEK mitotic genes were among the most significantly derepressed.These genes were deregulated to at least the same degree as cyclin A, a known target of the EF transcription factor.This method was used to identify novel EF target promoters, which we verified using two independent assays.Previous experiments have identified EF targets by profiling genes activated and repressed in response to ectopic expression of EF, EF, and EF. However, two potential limitations of such experiments are introduced by this approach.First, there is the potential for loss of target specificity resulting from overexpression.Second, this type of experiment does not permit a straightforward or unequivocal identification of direct targets, as secondary changes in gene expression resulting from progression through the cell cycle are possible.