Toutant M, Gauthier-Rouviere C, Fiszman M Y, Lemonnier M
Département de Biologie Moléculaire, C.N.R.S., Institut Pasteur, Paris, France.
Nucleic Acids Res. 1994 May 25;22(10):1838-45. doi: 10.1093/nar/22.10.1838.
The chicken beta tropomyosin (beta TM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the beta TM gene, we have analyzed the 5' regions associated with each CAP site. Truncated regions 5' to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (-168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5' to the beta TMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterized an enhancer element (-201/-68 nt) which contains an E box (-177), a variant CArG box (-104) and a stretch of 7Cs (-147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner.
鸡β-原肌球蛋白（β-TM）基因有两个可变转录起始位点（sk和nmCAP位点），分别在肌肉组织或非肌肉组织中使用。为了了解β-TM基因的组织特异性和发育调控表达所涉及的机制，我们分析了与每个CAP位点相关的5'区域。将nmCAP位点上游的截短区域插入细菌氯霉素乙酰转移酶（CAT）报告基因的上游，并将这些构建体转染到禽类成肌细胞和非成肌细胞中。在所有细胞类型中，最大转录由CAT构建体（-168 / + 216 nt）驱动。先前对β-TMskCAP位点5'区域的缺失分析表明，805 nt赋予肌管特异性转录。在这项工作中，我们鉴定了一个增强子元件（-201 / -68 nt），它包含一个E盒（-177）、一个变体CArG盒（-104）和一段7个C的序列（-147）。这些基序中的任何一个发生突变都会导致肌管特异性转录活性降低。电泳迁移率变动分析表明，这些顺式作用序列特异性结合核蛋白。这个增强子以方向依赖的方式发挥作用。
