Nucleotide sequence and transcriptional regulation of a positive regulatory gene of Shigella dysenteriae.

A 1,937 bp PstI-HindIII fragment containing the ipaR locus was cloned from the large invasion plasmid of Shigella dysenteriae CG097, and its nucleotide sequence was completely determined. The IpaR protein (35 kDa, calculated from the DNA sequence) was synthesized in Escherichia coli chi 1411 minicells containing the 1,937-bp PstI-HindIII fragment. To determine the regulatory role of ipaR for ipa genes, we applied genetic complementation experiments using chloramphenicol acetyltransferase (CAT) as reporter. Analyses of CAT activity of the recombinant plasmids containing the 5' flanking sequences of the 24-kDa-protein gene and the ippI, ipaB, ipaC, and ipaD genes defined strong promoters upstream of the 24-kDa-protein gene and ipaD gene, weak promoters upstream of the ippI and ipaB genes, and the absence of any promoter activity for the ipaC gene. Complementation analyses showed that the CAT activity only under direction of the ippI promoter region increased 1.8-fold in the presence of IpaR protein. On the basis of our data, we suggest that an operon comprising ippI, ipaB, and ipaC is positively regulated by IpaR protein which has a trans effect on a DNA sequence upstream of the ippI promoter.     

人类XBP1基因5′上游DNA序列的转录激活功能分析

目的分析人类转录因子X-盒结合蛋白1（X-box binding protein Ⅰ，XBP1）基因启动子在不同细胞系中转录活性的差别，研究其转录调控机制。方法在对XBP1基因进行生物信息学分析的基础上，设计6种XBP1启动子缺失突变体，分别包括剧XBP1基因5’上游-1039～66bp、-859～66bp、-623～66bp、-351～66bp，-227—66bp、-227～-45bp，针对每一种缺失突变体构建相应的氯霉素乙酰转移酶（chloramphenicol acetyltansferase，CAT）报告基因载体，再分别瞬时转染K562、HepG2、NIH-3T3和LO2细胞，应用报告基因CAT瞬时表达系统，检测XBP1每一种缺失突变体在K562、HepG2、NIH-3T3和LO2 4种不同细胞中的活性差异，并进一步确定脚，基因启动子的具体调节部位。结果成功获得XBP1基因启动子6种缺失突变体的正确克隆和6种相应的报告基因载体p1—XBP1p，p2-XBP1p，p3-XBP1p，p4-XBP1p，p5-XBP1p，p6-XBP1p，每一种报告基因转染K562、HepG2、NIH-313和LO2 4种不同细胞系后，p4-XBP1p和p5-XBP1p在K562、HepG2细胞中的转录活性均高于其它报告载体，p5-XBP1p在HepG2细胞中转录活性最高；而6种报告基因载体在NIH-3T3小鼠成纤维细胞中不具有转录活性。结论。XBP1基因的转录激活能力在不同细胞中有所差异，其表达活性具有一定的细胞类型特异性，与细胞类型和细胞状态密切相关；XBP1基因启动子的-227～66bp区域是该启动子的核心部位，而且核心启动子区包括ATG翻译起始密码子的下游部位，这一部位是转录活性的重要部位，一旦缺失，XBP1基因启动子会丧失活性。