Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.
Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.
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鉴定 L-CAM（一种钙依赖性细胞粘附分子）编码基因的启动子和转录增强子。
DOI：
10.1073/pnas.90.23.11356
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发表时间：
1993
影响因子：
11.1
通讯作者：
Edelman,GM
中科院分区：
文献类型：
--
作者：
Sorkin,BC;Jones,FS;Cunningham,BA;Edelman,GM
L-CAM是一种钙依赖性细胞粘附分子，在发育过程中以特征性的位置依赖性模式表达。以前的研究表明，在异源启动子的控制下，在转基因小鼠的鸡L-CAM基因的异位表达的顺式作用序列控制的时空表达模式的L-CAM基因本身存在。我们现在已经检查了L-CAM基因控制其表达的序列，并在基因的第二个内含子中发现了增强子。来自内含子的2.5-kb Kpn I-EcoRI片段作为猿猴病毒40最小启动子的增强子，驱动氯霉素乙酰转移酶（CAT）报告基因，并在MDCK细胞中对CAT活性产生14.0倍的诱导。为了缩小负责增强子活性的区域并确定增强子是否可以以细胞类型特异性方式起作用，在两种鸡细胞系中测试了来自内含子的许多较小的限制性片段的活性，所述两种鸡细胞系为产生高水平L-CAM的LMH肝细胞瘤系和产生很少（如果有的话）L-CAM的SL-29成纤维细胞系。四个L-CAM增强子质粒含有较短的片段来自内含子表现出增强的CAT活性水平（9.4和16.5倍之间），从转染的LMH细胞提取物，但不是从SL-29细胞。L-CAM增强子区域的DNA序列分析揭示了转录因子SP1、E2 A和AP-2的推定结合位点。此外，LE-9，最小的L-CAM增强子片段（310 bp），含有肝脏富集的POU同源结构域转录因子HNF-1的共有结合位点。上游序列的测试表明，一个630 bp的片段，对应于几乎整个基因间区之间的L-CAM和它的相邻CAM基因，K-CAM，可以作为一个启动子。与L-CAM增强子组合，该片段指导LMH细胞中CAT报告基因的细胞类型特异性表达，其水平与使用猿猴病毒40最小启动子的增强子构建体所观察到的水平相当。这些组合的观察定义了鸡L-CAM基因的启动子和增强子。他们提出的可能性，这些顺式作用的调控序列可能是在指导特定的地方依赖性表达的L-CAM基因在鸡。
L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.