摘要
目的检测正常人及视网膜母细胞瘤（ＲＢ）患者Ｒｂ基因５′端调控区的ＤＮＡ序列，以及不同的ＤＮＡ片段的转录调控活性，研究Ｒｂ基因启动子的结构、功能以及突变对功能的影响和与ＲＢ发病的关系。方法应用ＳＳＣＰ分析及ＤＮＡ序列测定检测正常人及ＲＢ患者Ｒｂ基因启动子的ＤＮＡ序列和点突变；分离Ｒｂ基因５′端不同位置及不同大小的ＤＮＡ片段，通过氯霉素乙酰基转移酶做报告基因，检测不同的ＤＮＡ片段的转录调控活性。结果Ｒｂ基因产物起始密码上游３２７ｂｐ至８７ｂｐ间２４０ｂｐ的ＤＮＡ片段具有基本的启动子功能，其上游和下游的ＤＮＡ序列内可能还存在正或负转录调控元件。１００例正常人Ｒｂ基因启动子ＤＮＡ序列未见有任何变异，但３０２例ＲＢ患者中５例有点突变并伴有ＣＡＴ活性明显降低。结论Ｒｂ基因启动子的ＤＮＡ序列非常稳定，其突变常常导致启动子活性下降。
Objective To investigate the DNA sequence of the 5′ untranslated region of the Rb gene in normal individuals and retinoblastoma patients and identify the elements involved in transcription regulation of Rb gene. Methods SSCP analysis and direct genomic DNA sequencing were used to identify variations or alterations in DNA of normal adult WBC and tumor DNA of retinoblastoma patients. The different DNA fragments located in the 5' untranslated region of Rb gene were amplified and inserted into an expression plasmid with a CAT reporter gene. The capacity of transcription regulation of isolated DNA fragments was determined by CAT assay. Results A 240 bp DNA fragment located between -327～-87 bp upstream of the first starting codon was shown to be the sequence with essential promoter function. The Sp1/RBF 1, ATF and E2F binding sites were present in this region and positive and negative elements were found further upstream and downstream. Four of 5 naturally occurring mutations were identified in either Sp1/RBF 1 or ATF binding sites from 302 patients with retinoblastoma. All these mutants had reduced CAT activity. However, 100 cases of normal adults did not show any variant DNA sequence in the same region. Conclusion The promoter DNA sequence of Rb gene is quite stable. Its variation usually leads to reduction in promoter activity which may be associated with genetic susceptibility to retinoblastoma.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
1998年第3期165-167,共3页
Chinese Journal of Oncology
关键词
视网膜母细胞瘤
RB基因
启动子
结构
基因突变
Genes, retinoblastoma Retinoblastoma Promoter regions(Genetics) Transcription, genetic Sequence analysis, DNA Point mutation
