The Cloning Of The Human DNA Repair Gene HR24L And The Study On Its Function

The subject of this study is to explore the function of human DNA repair gene. In this procedure, the open reading frame(ORF) of hR24L was amplified by PCR and was cloned into the retroviral expression vector pDor-neo; thus the sense and the anti-sense recombinant vectors were constructed. Then the human breast cancer MCF7 cells were transfected using this recombinants by lipofectin respectively and were scanned using G418, and thus the sense and the anti-sense hR24L transfected cell lines were obtained.The growth of MCF7 cells were observed and the expression of hR24L was monitored by Northern bloting for the purpose of exploring the effect of hR24L on cell cycle. As a result, the over-expression of hR24L in sense sequence transfected cells and its down-expression inanti-sense sequence transfected cells were observed. The former significantly suppressed the growth of MCF7 cells. Moreover, Cell cycle analysis by FACS indicated that G2/M arrest occured in the sense transfected cells, suggesting that hR24L protein was involved in the regulation of cell cycle control.To study the effects of the hR24L gene on the apoptosis of the transfected cells, H202, Sodium butyrate (NaBu)and serum starvation were used to induce apoptosis in the cells. FACS and DNA agaose gel electrophoresis were used for observing apoptosis. The results showed that hR24L was over-experssed in MCF7 cells transfected by sense recombinant vectors, while the expression was down-regulated in those cells transfected by anti-sense recombinant vectors. Moreover, both sense and anti-sense recombinant vectors inhibited the apoptosis caused by H202 and serum starvation, without any effects on the apoptosis caused by sodium butyrate. So it is concluded that the human repair gene hR24L is involved inthe regulations of apoptosis.Meanwhile, the ORF of hR24L from MCF7 cells was amplified by RT-PCR directly, the sequence of which was checked. After had been irradiated with gamma-ray at a total dose of 5 Gy and incubated for different period of time, the cells were collected and the total RNA was isolated. The analysis of hR24L-mRNA by Northern bloting indicated that the expression of hR24L-mRNA began to increase in about one hour after the radiation and reached the maximum in about two hours. It indicated that the expression of hR24L gene was damage-inducible, which can repair the damage caused by gamma-ray irradiation.