Cloning and characterization of the 5' flanking region of the stem cell factor gene in rat Sertoli cells.
Cloning and characterization of the 5' flanking region of the stem cell factor gene in rat Sertoli cells.
复制标题
大鼠支持细胞干细胞因子基因 5 侧翼区域的克隆和表征。
DOI：
10.1016/s0378-1119(96)00668-3
复制
发表时间：
1997
期刊：
影响因子：
3.5
通讯作者：
Boekelheide,K
中科院分区：
文献类型：
--
作者：
Jiang,C;Hall,SJ;Boekelheide,K
为了阐明大鼠睾丸支持细胞中干细胞因子（SCF，或钢因子/试剂盒配体）表达的分子基础，分离并表征了 SCF 基因 5' 侧翼区域的 1.5 kb。通过引物延伸测定和 cDNA 末端快速扩增 (RACE) 测定来鉴定转录起始点 (tsp)。在 tsp 上游 29 个碱基对 (bp) 处发现了一个 TATA 盒，并鉴定了许多转录因子共有序列，包括几个 AP2 和 Sp1 位点。使用瞬时转染原代大鼠支持细胞和其他 SCF 阳性和阴性细胞系的萤火虫荧光素酶编码基因 (luc) 表达载体，通过删除构建体分析 1.5 kb 5' 侧翼区域的转录活性。对于所有检查的细胞和细胞系，包括 tsp 的 -119 bp 至 +43 bp 片段足以满足 SCF 启动子活性，并且核心启动子活性不会因包含高达 -1461 bp 的上游序列而显着改变。这些结果表明需要该启动子区域之外的其他位点来定义 SCF 表达的细胞特异性调控元件。所有用环腺苷 3',5'-单磷酸 (cAMP) 和毛喉素处理的 SCF 缺失构建体的转录活性均增加了两到三倍，表明支持细胞中的 SCF 转录受到近端启动子区域中 cAMP 依赖性途径的调节。
In order to elucidate the molecular basis of stem cell factor (SCF, or steel factor/kit ligand) expression in Sertoli cells of rat testis, 1.5 kb of the 5′ flanking region of the SCF gene was isolated and characterized. The transcriptional start point (tsp) was identified by primer extension assay and a rapid amplification of cDNA ends (RACE) assay. A TATA box was found 29 base pairs (bp) upstream from the tsp, and a number of transcription factor consensus sequences, including several AP2 and Sp1 sites, were identified. The transcriptional activity of the 1.5 kb 5′ flanking region was analyzed by deletion constructs using a firefly luciferase-encoding gene (luc) expression vector transiently transfected into primary rat Sertoli cells and other SCF positive and negative cell lines. For all the cells and cell lines examined, a −119 bp to +43 bp fragment including the tsp was sufficient for SCF promoter activity, and the core promoter activity was not significantly changed by inclusion of upstream sequences as far as −1461 bp. These results indicate that additional sites outside of this promoter region are needed to define the cell-specific regulatory elements of SCF expression. The transcriptional activities of all SCF deletion constructs treated with cyclic adenosine 3′,5′-monophosphate (cAMP) and forskolin were increased two- to threefold, indicating that SCF transcription in Sertoli cells is regulated by a cAMP-dependent pathway in the proximal promoter region.