|Budget Amount *help
¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1995 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Rb is associated with the transcription factors such as E2F,MyoD and Myogenin, and its function is speculated to be an inhibitor of transcription factors. Transcription inhibitory activity of Rb is detected by CAT assay. However, hyperphosphorylation of Rb, i.e., inactivation of Rb, is observed during the whole period of DNA synthesis, suggesting that Rb regulates DNA synthesis. The purpose of this project is to learn wether or not Rb regulates DNA synthesis.
The promoter region of c-myc is known to contain the putative DNA replication origin. We transfected the plasmid having the promoter region of c-myc to the cell lines which express Rb or does not it. The copy number of myc was higher in the Rb negative cell line than the Rb co-transfected one. However, there was no difference in the copy number of myc between the Rb-negative and the positive cell lines in the plasmid which does not contain the promoter region of c-myc. These results indicate that Rb regulates the copy number of the
plasmid which contains the replication origin. Then, we tried to detect a replicated form of the plasmid to confirm that the diffrent copy numbers of the plasmid are due to the replication of the plasmid. The methylated plasmid is resistant to EcoRI,but when the plasmid replicated in cells, it become unmethylated and sensitive to EcoRI.When the methylated plasmid was transfected to the cells, Rb negative cells produced the unmethylated form of the plasmid but Rb positive cells did not. These results suggest that Rb was involved in replication of the transfected plasmid.
To confirm these results we tried to detect replication associated enzyme activity such as DNA helicase or DNA polymerase activity in the Rb immunocomplex in vitro. After the plasmid which contained the c-myc promoter region was added to Rb immunocomplex, the activity of helicase and DNA polymerase was assayd. As a result, no replication associated enzyme activity was detected in Rb immunocomplex even when the Rb was inactivated by phosphorylation with cdk2/cyclin A.Since, in vitro replication system of vertebrate cells has not been established except for SV40 replication origin at present. This system is not suitable for the present experiments, because it contains LT antigen which inhibits the function of Rb. In vitro replication systems other than the SV40 system must be developed to confirm that Rb is really involved in the replication of cells or not. Less