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首页> 外文期刊>Molecular and Cellular Biology >Basal promoter of the rat connexin 32 gene: identification and characterization of an essential element and its DNA-binding protein.
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Basal promoter of the rat connexin 32 gene: identification and characterization of an essential element and its DNA-binding protein.

机译：大鼠连接蛋白32基因的基础启动子：基本元素及其DNA结合蛋白的鉴定和表征。

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The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32-B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.
机译：连接蛋白32（Cx32）基因，一个多基因家族的成员，优先在肝脏中表达。大鼠Cx32基因的基础启动子复合体先前被定位在第一个外显子紧接上游的146-bp区域（图位[mp] -179至-34）。为了研究有助于基础启动子活性的生化因素，使用DNA迁移率变动分析研究了该区域（mp -177至-106）内的核蛋白-DNA复合物。用来自肝癌细胞系HuH7和FAO-1的核蛋白提取物鉴定了三种DNA-蛋白质结合活性，分别称为Cx32-B1，Cx32-B2和Cx32-B3。但是，在正常大鼠肝脏组织的核蛋白提取物中仅检测到Cx32-B2结合活性。在大鼠肝组织中，这种活性明显比在肝癌细胞系和其他器官中的组织丰富。通过使用甲基化干扰足迹法，将Cx32-B2复合物定位于mp -152和-127之间的区域，并在大鼠肝核提取物中将DNA探针包含与60-kDa蛋白结合的区域。 Cx32-B2结合位点中的两个核苷酸的突变消除了Cx32-B2蛋白质-DNA复合物的形成，并显着降低了Cx32启动子的转录活性。这些结果表明，Cx32-B2复合物是大鼠Cx32基础启动子的重要组成部分，可能是该基因在肝脏中优先表达的主要因素。

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