Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor
Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor
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DOI：
10.1074/jbc.273.34.21594
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发表时间：
1998-08-21
影响因子：
4.8
通讯作者：
Johnson, AC
中科院分区：
文献类型：
--
作者：
Reed, AL;Yamazaki, H;Johnson, AC
gc结合因子（GCF）抑制某些基因的转录，并由3.0千碱基mRNA编码（Kageyama， R., and Pastan， I. (1989) Cell 59, 815-825）。GCF cDNA与另外两个mRNA物种杂交，分别为4.2和1.2千碱基。我们使用差分杂交技术鉴定了4.2千碱基mRNA的cDNA克隆（称为GCF2），发现它在HUT-102细胞中高度表达。开放阅读框由2256个核苷酸组成，编码一个含有752个氨基酸的蛋白质，计算分子质量为83千道尔顿。GCF2在体外用网状细胞裂解物表达，大肠杆菌在sds -聚丙烯酰胺凝胶电泳中以160千道尔顿的蛋白迁移，但在质谱分析中其分子质量为83千道尔顿。GCF2与表皮生长因子受体启动子片段结合，主要结合位点位于核苷酸-249和-233之间。共转染实验表明，GCF2在含有GCF2主要结合位点的构建体中抑制表皮生长因子受体启动子的转录，而在该位点发生突变时则不抑制。因此，GCF2是一种新发现的具有异常电泳迁移率的转录抑制因子。
GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825). The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases. We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4.2-kilobase mRNA and find that it is highly expressed in HUT-102 cells. The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons. GCF2 expressed in vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis. GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides -249 and -233. Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated. Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility.