Amongst the inadequate inhibitors recognized there is a distinct differential sample

It has been shown that the S phase duration of serum-deprived HepG2 cells is about 11-29 h after serum stimulation. Our data showed that the luciferase activity of wild-type P1 markedly increased at 12 h and peaked at 20 h after serum stimulation, suggesting an S phase-specific induction; however, this effect was less pronounced for the mE2F construct. Taken together, our results indicate that the preferential expression of R2 gene in proliferating cells is associated with S phase-specific P1 activation that results from the relief of E2Fmediated repression. Through evolution, expression of RNR subunit genes is tightly controlled in response to DNA damage and their transcripts from multiple promoters or alternative splicing often exhibit distinct physiological implications. To determine whether and which transcript variant of R2 gene in zebrafish is induced by DNA damage, expression of R2 gene in developing embryos treated with DNA damage reagents was investigated using realtime PCR. As shown in Figure 6A, treatment of developing embryos with 2 000 or 4 000 nM Camptothecin led to a 3- to 13-fold increase in the levels of four R2_v3 transcripts that are derived from P3 promoter. Levels of R2_v3c and R2_v3d increased 11- and 13-fold, respectively; however, R2_v1&2 levels were nearly unaffected. To further determine whether DNA damage reagent could induce expression of R2_v3 at the level of transcription, luciferase activity of pGL-(-5194/-954) in CPTtreated embryos was tested. As shown in Figure 6B, the activity of P3 was induced by certain concentrations of CPT in a dosedependent manner. These results suggest that R2_v3 transcripts are specifically induced by DNA damage signals and that this inductive effect is closely associated with the transcriptional activation of P3. Since subcellular distribution of RNR subunits play crucial roles in the regulation of RNR activity, we investigated the localization of the three putative R2 isoforms in transfected Hela cells. As previously described, the coding sequences of three R2 isoforms and R1 were tagged with Flag, HA, GFP or RFP. Immunofluorescence staining assays indicated that three isoforms of zebrafish R2 were mainly distributed in the cytoplasm of Hela cells. Moreover, GFP-tagged R2 and RFP-tagged R1 were co-localized in the cytosol of Hela cells. Next, we addressed whether N-terminally truncated R2 isoforms are able to associate with R1. HA-tagged R1 and one of the Flag-tagged R2 isoforms were co-expressed in transfected HEK293T cells. Co-immunoprecipitation and Western blotting assays were then conducted with monoclonal antibodies against Flag or HA. As shown in Figure 9, D29R2 and D52R2 can be precipitated with HA-tagged R1 and detected using the anti-Flag antibody, while R1 can be precipitated with either Flag-tagged D29R2 or D52R2 and detected using the anti-HA antibody. These results suggest that N-terminally truncated isoforms of zebrafish R2 are able to physically interact with R1. RNR subunits are highly conserved during evolution and their expression is tightly controlled by multiple mechanisms. However, it remains largely unknown about regulation and functions of RNR subunits in zebrafish. A transcript encoding the normal form R2 in zebrafish has been identified without characterization of its functions. We have recently shown that expression and functions of p53R2 in zebrafish are closely associated with its activities in DNA repair and synthesis. In this study, we demonstrate intrinsic mechanisms underlying the control of zebrafish R2 expression, including alternative promoter usage, pre-mRNA splicing and polyadenylation site selection. Six distinct transcripts that are derived from three promoters are characterized to encode three R2 isoforms. Transcripts of normal R2 is mainly expressed in a cell cyclespecific manner, while transcripts of D29R2 and D52R2 are induced by DNA damage. Our results provide new evidence for the tight control of differential expression and functions of R2.