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目的 克隆低剂量辐射诱导基因 ,研究辐射对其转录调控作用和生物学功能。方法用mRNA差异显示技术分离辐射诱导表达基因 ,用RACE技术获取cDNA旁侧序列 ,Northern杂交分析基因的转录调节 ,生物信息学分析基因的结构和功能。结果 获得包括 3’端在内的辐射诱导新基因LRIGx的cDNA片段 ,序列同源性比较显示与人染色体 2 0q11 2 12一段DNA高度同源 (>99 % )。Northern杂交结果揭示该基因转录子全长约 8 5kb ,在 0 2Gy照射后 2h出现诱导表达 ,4h后转录子水平为对照细胞的 5倍多 ,也受 0 0 2Gy更低剂量照射诱导表达。 2Gy大剂量照射后 1h出现短暂诱导表达 ,但不如低剂量照射明显 ,2h后恢复正常水平。生物信息学分析结果显示该基因编码产物含有解旋酶活性保守区。结论 分离鉴定出一低剂量辐射反应基因 ,其编码蛋白可能参与DNA代谢 (如修复 )等细胞辐射反应过程。
Objective To clone a low-dose radiation-induced gene and study its transcriptional regulation and biological functions. Methods The mRNAs of differentially expressed genes were isolated by mRNA differential display. The cDNAs were sequenced by RACE. The transcriptional regulation of the genes was analyzed by Northern blotting. The structure and function of genes were analyzed by bioinformatics analysis. Results A cDNA fragment of the LRIGx gene, including the 3 ’end, was obtained. A comparison of sequence homologies showed that it was highly homologous (> 99%) to the DNA of human chromosome 20q11-212. The results of Northern blotting revealed that the full length of this gene was about 85kb. The expression was induced 2h after 0 2Gy irradiation. After 4h, the transcript level was more than 5 times that of control cells, and was also induced by lower dose of 0 2Gy. 2Gy high-dose irradiation after 1h appeared transiently induced expression, but not as good as low-dose irradiation, returned to normal after 2h. Bioinformatics analysis showed that the gene encoding product contains conserved regions of helicase activity. Conclusion A low dose radiation response gene was isolated and identified. The encoded protein may be involved in the cellular radiation response such as DNA metabolism (eg, repair).