Effect Of Human Elongator Subunit Elp3 And Human Transcription Factor TFIIF Subunit RAP30 On 293T Cell Growth And Transcription Function

Objective: To construct Elp3-293T and RAP30-293T cell lines which stably expressed Elp3 and RAP30.Study the effect of Elp3 and RAP30 on the cell growth and transcription activtiy of 293T.Methods: Transfected the recombinant eukaryotic expression plasmids pFLAGCMV 4-Elp3 and pFLAGCMV4-RAP30 into human embryo kidney fibroblasts cell line 293T by Lipofectamine reagent respectively. Cell clones which stably expressed Elp3 or RAP30 were obtained in the medium which contained G418 after 3-4 weeks. Objecive genes were detected by RT-PCR, indirect immunofluorescence. MTT assay and Flow cytometry were applied to measure the effect of Elp3 and RAP30 on cell growth and cellcycle of 293T. We also observe the effect of Elp3, Elp4 and RAP30 on transcription activity by Luciferase Reportor Gene Assay.Results: Result of RT-PCR and indirect immunofluorescence showed that Elp3 gene could stably express in 293T cells.MTT assay showed that Elp3 inhibited cell growth of 293T. Result of Flow cytometry demonstrated that Elp3 can induce 293T cell delayed in G1 phase of the cell cycle,but can not induce cell apoptosis.Results of luciferase activity assay showed Elp3 can active transcription. Elp3 and Elp4 can synergistically active transcription. The recombinant eukaryotic expression plasmid pFLAGCMV4-RAP30 was confirmed by PCR and endonucleases digestion. The full cDNA sequence of RAP30 was agree with Genbank. Result of RT-PCR demonstrated RAP30 gene could express in 293T cells. Results of Flow cytometry demonstrated that RAP30 can induce 293T cell delayed in G2 phase of the cell cycle. RAP30 can active transcription. RAP30 and Elp3 can synergistically active transcription.Conclusion: Overexpression of Elp3 can inhibit the cell growth of 293T induce cells delayed in G1 phase of the cell cycle.It can weakly active transcription and synergistically active transcription in the presence of Elp4. RAP30 can induce 293T cell delayed in G2 phase of the cell cycle. RAP30 can active transcription. RAP30 and Elp3 can synergistically active transcription.