Chang M H, Ng C K, Lin Y J, Liang C L, Chung P J, Tyan Y S, Hsu C Y, Shu C H, Chang Y S
Graduate Institute of Basic Sciences, Chang-Gung College of Medicine and Technology, Taoyuan, Taiwan, ROC.
DNA Cell Biol. 1997 Jul;16(7):829-37. doi: 10.1089/dna.1997.16.829.
Latent membrane protein 1 (LMP 1) is one of two Epstein-Barr virus (EBV)-encoded proteins that expressed in nasopharyngeal carcinoma (NPC) cells. Previous studies showed that a 3.5-kb transcript of the LMP 1 gene, in addition to the 2.8-kb transcript, was detected in a B95-8-EBV-containing, nude mice-passaged NPC tumor, C15. This indicated that a transcript was initiated from a region 5' to the putative promoter, ED-L1. We have isolated an EBV variant from a NPC tissue, and this virus strain contained a more pathogenic LMP 1 gene. DNA sequence analysis of the 5'-upstream region showed distinct variations as compared to that of B95-8 strain. To test if the LMP 1 gene of the NPC strain also contained an upstream promoter, we generated a series of deletion plasmids encompassing positions -1,030 to +20 of the LMP 1 promoter and tested for their abilities to drive the expression of the reporter gene in human epithelial cell lines, C-33A and NPC-TW076. We found that the region between -643 and -496 contained a promoter activity that was approximately five-fold higher than the putative promoter, ED-L1. This region between -643 and -496 was designated as ED-L1E. C-33A cells containing the genomic clone pT7(E) or the clone that had deleted a 94-bp ED-L1 sequence (delta94) was used to determine the transcription initiation sites by RNase protection assay. Results showed that a transcription initiation site was located at nucleotide 170,099 ("A") of EBV genome. The transcript was expressed in NPC biopsies and in human primary normal epithelial cells transfected with pT7(E) and delta94, respectively, as examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Furthermore, the ED-L1E was not regulated by the EBV-encoded nuclear antigen 1-mediated transcriptional enhancer family of repeats (FR) in C-33A cells. Our results suggested that the ED-L1E was specifically activated in epithelial cells. The biological significance of the selective usage of the ED-L1E promoter was discussed.
潜伏膜蛋白1（LMP 1）是在鼻咽癌（NPC）细胞中表达的两种爱泼斯坦-巴尔病毒（EBV）编码蛋白之一。先前的研究表明，在一只含有B95 - 8 - EBV、经裸鼠传代的NPC肿瘤C15中，除了检测到LMP 1基因的2.8 kb转录本外，还检测到了一个3.5 kb的转录本。这表明一个转录本是从假定启动子ED - L1上游的一个区域起始的。我们从一个NPC组织中分离出了一种EBV变体，该病毒株含有一个致病性更强的LMP 1基因。对5' - 上游区域的DNA序列分析显示，与B95 - 8株相比存在明显差异。为了检测NPC株的LMP 1基因是否也含有一个上游启动子，我们构建了一系列缺失质粒，其包含LMP 1启动子从 - 1030到 + 20的位置，并检测它们在人上皮细胞系C - 33A和NPC - TW076中驱动报告基因表达的能力。我们发现 - 643和 - 496之间的区域含有一种启动子活性，其比假定启动子ED - L1高约五倍。 - 643和 - 496之间的这个区域被命名为ED - L1E。通过核糖核酸酶保护试验，使用含有基因组克隆pT7(E)或缺失了94 bp ED - L1序列的克隆（delta94）的C - 33A细胞来确定转录起始位点。结果显示转录起始位点位于EBV基因组的核苷酸170,099（“A”）处。通过逆转录 - 聚合酶链反应（RT - PCR）分析检测到，该转录本分别在NPC活检组织以及用pT7(E)和delta94转染的人原代正常上皮细胞中表达。此外，在C - 33A细胞中，ED - L1E不受EBV编码的核抗原1介导的转录增强子重复序列家族（FR）的调控。我们的结果表明ED - L1E在上皮细胞中被特异性激活。文中讨论了选择性使用ED - L1E启动子的生物学意义。
